CN1768138A - Methods of producing recombinant proteins in microorganisms - Google Patents

Abstract

The present invention discloses a method for producing recombinant functional plasminogen in a microorganism and a method for identifying a plasminogen activator. A nucleic acid sequence encoding a functional part of plasminogen is fused with a nucleic acid sequence encoding at least a signal peptide. The nucleic acid molecule encoding the functional plasminogen and the nucleic acid molecule encoding the signal peptide are coupled by a protease cleavage site codon, which ensures the cleavage of the signal peptide. Appropriate application of a preparation containing the recombinant plasminogen or the corresponding plasmin can treat slow-healing or refractory wounds.

Description

Methods of producing recombinant proteins in microorganisms

technical field

The present invention relates to methods for the production of recombinant proteins in microorganisms. More specifically, the present invention relates to methods of producing recombinant functional plasminogen in microorganisms and methods of identifying plasminogen activators.

Background technique

The human fibrinolytic system contains a core component—fibrinolytic enzyme (Pm, hereinafter referred to as plasmin). Plasmin can degrade fibrin on the one hand, and activate matrix metalloproteinases (MMPs) and growth factors on the other hand, and activated matrix metalloproteinases (MMPs) and growth factors play a joint role in the degradation of extracellular matrix and wound healing.

Plasmin comes from its precursor molecule—plasminogen (hereinafter referred to as plasminogen). So far, two physiological activators of plasminogen (also called plasminogen activators, PA) are known. These are tissue-type plasminogen activator (tissue-type PA, t-PA) and urokinase-type plasminogen activator (urokinase-type PA, u-PA). In addition, this system is regulated by a suite of protease inhibitors such as α2-antiplasmin. Plasminogen and plasmin have very important biological functions, which are directly related to two different activators.

The so-called t-PA-mediated pathway is responsible for regulating fibrin homeostasis in vivo, while the u-PA-mediated pathway is prominent in cell motility and tissue remodeling. This function was particularly prominent when u-PA-deficient mice suffered from chronic, non-healing wounds. Mice with inactivated genes for plasminogen, t-PA, and u-PA developed the same disease. In addition, the life span of experimental animals is significantly shortened, especially due to thrombosis and organ failure. An overview of the plasminogen/plasmin system has been described in a book published by Desire Collen ("Thrombosis and Haemostasis", 82, 1999 (1)).

Plasmin therapy is indicated in the treatment of heart attack or stroke patients, where rapid dissolution of fibrin clots is necessary for survival, therefore, said plasmin therapy is in addition to the use of plasminogen activators In addition to treating patients, plasminogen activators only indirectly contribute to the hydrolysis of fibrin clots.

The mouse disease model mentioned above shows that plasmin is also a potential therapeutic agent for wounds that do not heal or only slowly heal.

Usually t-PA activates plasminogen only in the presence of fibrin, that is, after the coagulation cascade of blood is completed. In the absence of substrate, plasmin is always immediately inhibited by α2-antiplasmin. It is recognized that this inhibitory effect is significantly attenuated when plasmin binds to fibrin and thereby activates fibrin clot lysis.

Since dissolving blood clots is often necessary for patient survival in the case of heart attack or stroke, different plasminogen activation protocols are used in the treatment. For example, injection of streptokinase allows rapid recanalization of blood vessels. Therefore, streptokinase activates plasminogen, not on the basis of proteolytic activation, but forms a complex, which then activates other plasminogen molecules to convert them into plasmin, so Streptokinase is a bacterial protein.

Further research on the therapeutic use of urokinase concluded that, like streptokinase, urokinase cannot at the molecular level distinguish between plasminogen bound to fibrin and free plasminogen. Therefore, recombinant human t-PA was developed, and in clinical research, it has been confirmed that recombinant human t-PA is superior to streptokinase. However, this diagnostic finding has not been confirmed by other studies.

The production system established by the method of molecular genetics plays an important role in the production of recombinant proteins used in modern treatments, and recombinantly produced plasminogen activators such as rt-PA (and its different derivatives), recombinant Single-chain urokinase-PA and recombinant staphylokinase again just highlight the importance of the production system.

Plasminogen is a precursor molecule of fibrinolytic enzymes. The cDNA of human plasminogen has been published in the scientific literature (Malinowski et al., Biochemistry, 23, 1984 (12); Forsgren et al., FEBS Lett. 213, 1987 (2)) and a gene containing a non-coding intron ( Petersen et al., J. Biol. Chem., 265, 1990(3)).

Human plasminogen (hPg) is the zymogen of plasmin as a serine protease, which is a glycoprotein composed of a polypeptide chain of 791 amino acids, with a molecular weight of 92,000, a theoretical isoelectric point of 7.1, and a glycosylation ratio of 2% (Collen, 1999, (1)). Plasminogen is produced in the liver at a plasma concentration of approximately 200 mg/l [1.5-2 μM (micromoles per liter)].

This molecule is divided into seven structural domains, including the N-terminal pro-activating peptide (Glu-1～Lys-77), the partially homologous five ring cake domains (Kringle domain) and the protease domain (Val -562 to Asn-791; Collen, 1999(1)). The catalytic triad structure is consistent with that of all serine proteases and consists of the following amino acids: His-603, Asp-646 and Ser-741. Ring cake domain 1 is the recognition sequence for plasminogen binding to fibrin (Petersen et al., 1990 (3)) and for binding of plasminogen to various cell surface receptors.

Among the post-translational modifications, two essential glycosylation sites, Asn-289 and Thr-346, are located in the ring cake domain 3, which is particularly important for the function of plasminogen (with different proteases and streptokinases, respectively). activating ability and also has receptor binding properties). Depending on the modifications described, two main forms of plasminogen are distinguished:

- Plasminogen I has the above-mentioned glycosylation properties

- Plasminogen II lacks the Asn-289 glycosylation modification.

Another glycosylation site is amino acid Ser-248. Amino acid Ser-578 may exist in phosphorylated form.

In vivo, plasminogen is activated by proteolytic cleavage between amino acids Arg-561 and Val-562. Subsequently, hydrolytic activation of another proteolytic enzyme occurs between Lys-77 and Lys-78 to form Lys-78-hPg. Alternatively in Glu-Pg, the chemical bond can also be directly hydrolyzed first. The activated plasmin Lys-78-hPm are all linked by disulfide bonds. Thus, the heavy chain of hPm (1/78-561) is responsible for interacting with substrates such as fibrinogen and fibrin. The light chain (562-791) derived from the C-terminus is the catalytically active subunit.

It is known from the literature that the recombinant production of the fibrin-binding domain of plasminogen in Pichia pastoris yields 17 mg/l (Duman et al., Biotechnol Appl Biochem. 28; 39 -45, 1998(4)). The authors could confirm the glycosylation sites of this domain (ring cake 1-4). Another citation describes the production of two domains, ring cake 4 and 5, of human plasminogen (Guan et al., Sheng Wu Gong Cheng Xue Bao, 17, 2001(5)). The aim of this work was to identify domains capable of inhibiting endothelial cell growth.

However, none of the plasminogen domains produced recombinantly in Pichia pastoris by the above two working groups contained the catalytic domain that is decisive for physiological functions.

Gonzalez-Gronow et al. (Biochimica et Biophysica Acta, 1039, 1990 (6)) compared the expression of recombinant human plasminogen in Escherichia coli and COS cells (a simian kidney cell line) with each other. Microbial production in E. coli was a failure, which the authors attribute to insufficient glycosylation. Peptide chains could be successfully produced, however in a non-activatable form, ie after treatment with activators (urokinase and t-PA), no activated plasmin could be produced.

Lack of glycosylation of proteins results in defects in important physiological functions related to activation capacity (no detectable enzymatic activity) and to endothelial cell recognition (Gonzalez-Gronow et al., Biochimica et Biophysica Acta, 1039, 1990(6)). Furthermore, post-translational modification with carbohydrates has a dramatic effect on the half-life of proteins in mammalian blood.

However, the authors produced functional plasminogen in COS cells. Other authors describe functional expression in insect cells (Whitefleet-Smith et al., Arch. Biochem. Biophys., 271, 1989 (7)). However, when mammalian cells and insect cells are used, there are disadvantages of wasted time, high cost of culture conditions and low yield of protein obtained. In addition, in mammalian cells, due to the intracellular expression of proteins, the cytoplasm contains proteases, so it is not suitable for the production of more zymogens [Nilsen and Castellino, Protein Expression and Purification (Protein Expression and Purification), 16 , 1999(8); Busby et al., J. Biol. Chem., 266, 1991(9)]. Usually in the baculovirus/lepidopteran (insect cell) system, the expression yield of this system is 3-10 mg/ml.

In the patent application with publication number WO0250290, a method for recombinantly producing functional mini-plasminogen and micro-plasminogen in yeast is disclosed. To this end, the authors expressed either the catalytic domain gene (small plasminogen) containing the ringcake domain or human plasminogen without the ringcake domain in Pichia pastoris as host organism (microplasminogen). The thus recombinantly produced miniplasminogen and microplasminogen were subsequently purified and processed into miniplasmin and microplasmin, respectively, and their activities were demonstrated through animal experiments. The yield of recombinant protein was stated to be 100 mg/l for small plasminogen and 3 mg/l for micro plasminogen. However, it is more difficult to achieve higher expression of recombinant protein products, as demonstrated in patent application publication number WO0250290, in which the production of small plasminogen and microplasminogen Significantly decreased by 2 orders of magnitude. No mention is made of any examples of embodiments expressing long-segment plasminogen variants such as Lys-plasminogen or glutamate-plasminogen (Glu-plasminogen).

Methods for the recombinant production of functional plasminogen in microorganisms have not been disclosed to the extent that a person skilled in the art can perform them.

Contents of the invention

Therefore, the object of the present invention is to produce functional human plasminogen in a cheap way and further process it into plasmin with catalytic activity.

As claimed in claim 1, the method for the recombinant production of plasminogen in microorganisms achieves said object. Further solutions are mentioned in the independent claims. The dependent claims reflect preferred embodiments.

It was a surprising discovery that it is possible to produce recombinant functional glutamate plasminogen or lysine plasminogen in microorganisms. Further research on this basis showed that recombinant microplasminogen, small plasminogen, lysine plasminogen and glutamate plasminogen could achieve unexpectedly high expression levels.

The subject of the present invention is the use of molecular genetics methods to clone plasminogen genes in expression vectors, preferably microplasminogen and small plasminogen genes, more preferably glutamic acid A plasminogen or lysine plasminogen gene, or a functional variant of each of the foregoing genes; and recombinantly produced functional plasminogen, preferably functional human plasminogen. Furthermore, the present invention describes a method for the identification of proteases that catalyze the conversion of plasminogen to plasmin. The plasminogen and plasmin produced by the present invention, respectively, will not be contaminated by animal proteins or viruses, which usually occur during isolation from humans, cattle and other mammals and can cause patient adverse reactions.

The present invention is characterized by a method for producing a recombinant protein, the method comprising at least the following steps: a) fusing a nucleic acid sequence encoding at least a functional part of the plasminogen peptide with a nucleic acid sequence encoding at least one segment of signal peptide, for cutting The signal peptide, the nucleic acid sequence encoding the functional plasminogen peptide and at least the nucleic acid sequence encoding the signal peptide are coupled by the codon of the enzymatic cleavage site of the protease. Recombinant production systems are increasingly producing therapeutic protein products. Due to cost factors, efforts should be made to produce such recombinant products in microorganisms, especially bacteria. Said system has the following advantages: in addition to the relatively low price of the product, the protein yield can reach g/l level, and the recombinant protein will not be contaminated by viruses or proteins like prions that are harmful to patients. Since bacterial production systems are often unable to produce correctly folded proteins, production is often performed in eukaryotic cell systems, such as yeast, insect cells, or mammalian cells, in addition to in vitro refolding of misfolded proteins. cell. Eukaryotic cell strains and cell lines for production offer the advantage that they can be used to produce glycosylated proteins. But especially recombinant protein products derived from insect cells or mammalian cells are extremely costly and often have very low yields. In addition, they have the disadvantage of being contaminated with viruses and proteins that are harmful to humans. Said contamination does not occur with the use of eukaryotic microorganisms. The equipment required for eukaryotic microbial culture is similar to that required for bacterial culture, and there will be no contamination by mammalian viruses and proteins, and the protein yield can reach g/l level. A particularly suitable eukaryotic host organism is a branch of yeast, preferably Ascomycota. More preferred are Saccharomycotina, especially the class Saccharomycetes, with the order Saccharomycetales being particularly important. According to a more advantageous embodiment, the host should be of the family Saccharomycetaceae, especially of the genus Pichia. Preferred exemplary eukaryotic microorganisms of the present invention are Saccharomyces cerevisiae commonly used by bakers, others such as Candida, Pichia pastoris belonging to methanolotrophic yeast, methanolotrophic yeast Pichiamethanolica and Hansenula polymorpha, or filamentous fungi of the genus Aspergillus such as Aspergillus niger, Aspergillus oryzae and Aspergillus nidulans. Particularly preferred is Pichia pastoris.

Another feature of the production method of the recombinant product is that the nucleic acid molecule encoding at least the functional part of plasminogen is integrated into the expression vector used in these microorganisms, these nucleic acid molecules preferably encoding human plasminogen, and encoding at least A nucleic acid molecule fusion of a signal peptide [preferably a prepropeptide, preferably used for transport to the endoplasmic reticulum]; a codon for a protease cleavage point is inserted between the two nucleic acid molecules, which can be cleaved in the host Signal sequence or propeptide. A nucleic acid molecule preferably used is a nucleic acid molecule encoding human plasminogen. In addition to nucleic acid molecules encoding human plasminogen, those encoding plasminogen derived from other mammals may also be used. In this way, various mammalian plasminogens can be produced. Recombinant human plasminogen can be formed by overexpression according to the method of the present invention and, if desired, can be secreted into the culture medium and subjected to centrifugation, filtration or precipitation and incubation according to methods known to those skilled in the art. The host cells in the medium can be separated and the protein can be purified without complicated cell lysis process. Plasminogen is activated to plasmin by a protease that has the ability to process plasminogen into catalytically active plasmin.

Definitions of the following terms used in the present invention are:

By "recombinant production method" is meant the expression of a nucleic acid sequence (preferably a DNA sequence) derived from a clone, as well as fusion products of individual nucleic acid fragments, in a suitable host to produce a peptide or protein.

The "cloning" mentioned herein shall include all known cloning methods in the prior art. However, the methods will not be described in detail since they belong to common techniques obvious to those skilled in the art.

The "expression in a suitable expression system" mentioned herein shall include all known expression methods in the prior art, especially the expression methods mentioned in the claims.

In the term "functional plasminogen peptide part", it should be understood as a part of plasminogen or plasminogen-peptide that can complete the biological functions related to plasminogen. These relevant biological functions have at least the ability to be activated into plasmin by plasminogen activators, such as tissue plasminogen activator, urokinase, vampire-bat fibrinolysis Enzyme activator, streptokinase, staphylococcal kinase (hereinafter referred to as staphylokinase) and Pla protein derived from Yersinia pestis (Yersinia pestis), etc.; and proteolytic ability, characterized in hydrolyzing fibrin. The term "plasminogen activator" used in this specification and examples shall refer to plasminogen activators that can undergo proteolysis and those that cannot.

Furthermore, glutamate plasminogen is to be understood as meaning glutamate plasminogen which has the ability to be processed into lysine plasminogen by plasmin-catalyzed cleavage of the preactivation peptide.

After plasminogen binds to fibrin, laminin, fibronectin, hyaline junction protein, heparan sulfate glycoprotein, type 4 collagen and other substrates, its activation ability can be increased to 1000 times, and the same, the activation Capabilities are biological functions.

It should be understood that plasmin obtained after processing plasminogen should ensure plasmin-related biological functions, including the degradation of: laminin, fibronectin, hyaline junction protein and heparan sulfate glycoprotein; Activation of: procollagenase, promatrix metalloproteinase, potentially macrophage elastase, prohormones, and growth factors such as TGF beta-1 (potential transforming growth factor), VEGF (vascular endothelial growth factor) or bFGF (Basic Fibroblast Growth Factor).

Another biological function of plasmin is the ability to be inhibited by plasmin inhibitors, such as α ₂ -antiplasmin and α ₂ -macroglobulin.

According to the relevant biological functions of plasmin, its functions include not only binding fibrin, laminin, fibronectin, hyaline junction, heparan sulfate glycoprotein and type 4 collagen, but also binding receptors such as α-ene Alcoholase, annexin II or amphoterin.

Initially, all plasminogen formed is the inactive glutamate plasminogen form. This glutamic acid plasminogen can be cleaved by plasmin to generate lysine plasminogen. Both can be proteolytically cleaved by tissue plasminogen activator (in this case, only the aforementioned proteolytic activator) to generate plasmin, which consists of disulfide-linked subunits. The smaller subunit includes a proteolytic domain and a phosphorylation site, and the larger subunit carries three glycosylation sites and is responsible for binding to fibrin. The importance of glycosylation sites is further reflected in the stability in plasma. By forming a 1:1 complex with streptokinase or staphylokinase, plasminogen can be converted into an enzyme with proteolytic activity, which can process plasminogen into plasmin.

Accordingly, functional plasminogen refers to a plasminogen capable of being acted upon by a plasminogen activator to become plasmin with proteolytic activity. Further, the functional plasminogen preferably includes a fibrin binding domain, and may include at least one of three glycosylation sites.

The smallest forms of functional plasminogen are microplasminogen and small plasminogen, and the slightly larger form is lysine plasminogen. Glutamate plasminogen, which still includes the proactivator peptide, is also functional plasminogen. It is conceivable, however, that said regions can be neglected, especially regions present in long chains without significantly hindering said functions, especially proteolytic functions, fibrin binding functions.

It will be apparent to the skilled person to make different forms of plasminogen comprising a functional catalytic domain (hereinafter referred to as plasminogen derivatives). Insofar as the functions have been described, it is understood that said plasminogen variants have proteolytic properties after activation by plasminogen activators, such as streptokinase or urokinase.

- The catalytic domain may include, deleted and substituted amino acids, or be fused to other amino acids or to peptides or proteins.

- The large domain can include all intermediate regions from Glu20 to Arg580 (based on the preplasminogen sequence), which can be activated by plasminogen activators to generate activated plasmin.

The following are specific examples of the three forms of lysine plasminogen:

Variant 1: N-terminal amino acid: Met88

Variant 2: N-terminal amino acid: Lys97

Variant 3: N-terminal amino acid: Val98

Preferably, the plasminogen derivative is about 1 to 50 amino acids shorter or longer than the corresponding micro-, small-, Lys- or Glu-plasminogen; or is preferably characterized by a substitution of 1 to 10 amino acids, in addition , said derivative exhibits the property of being activated by a plasminogen activator. Between these specific micro-, small-, Lys- or Glu-plasminogens and the corresponding plasminogen derivatives, the sequence homology (sequence matching) exceeds 80%, preferably exceeds 85%, more preferably More than 90%, more preferably more than 95%, particularly preferably more than 98%, even more preferably more than 99%.

Preferred plasminogen derivatives have the following characteristics:

- the catalytic domain may comprise, at least one deletion, and/or a substitution of at least one amino acid, and/or a fusion with at least another amino acid or at least another peptide or at least another protein;

- The large domain can contain all the intermediate region from Glu20 to Arg580 (based on the preplasminogen sequence), which can be activated by plasminogen activators to generate activated plasmin;

- Derivatives of plasminogen, characterized by an amino acid sequence homology (sequence matching) of more than 80%, preferably more than 85%, more preferably more than 90%, still more preferably more than 95%, still more particularly preferably more than 99% .

"Microorganisms" include all life forms that possess the characteristics of tiny individuals. Thus, it includes both eukaryotic and prokaryotic microorganisms. Specifically refers to bacteria, yeasts, fungi and viruses.

"Nucleic acid" includes DNA and RNA, both in all possible configurations, eg, nucleic acids in double-stranded form, nucleic acids in single-stranded form, combinations thereof, and linear or circular nucleic acids.

It should be understood that "signal sequence" is a sequence of a peptide chain, which can ensure that another peptide chain sequence enters or crosses a biomembrane, such as entering the endoplasmic reticulum. Thus, examples of the signal sequence may be prepropeptide, prepeptide or propeptide.

"Cleavage point" means a site in a peptide sequence that provides a site for processing in a host organism to: cleavage of a signal sequence, pre-propeptide or propeptide from another peptide sequence, Or usually cut a peptide sequence into two parts.

"Nucleic acid encoding at least one signal peptide or propeptide" refers to a nucleic acid sequence that encodes a peptide or protein structure that facilitates the passage of other polypeptides across biological membranes, eg, into the endoplasmic reticulum.

"Primer" refers to a starting oligonucleotide. Means a short, single-stranded, oligoribonucleic acid or deoxyribonucleic acid that is complementary to a region of a single-stranded nucleic acid molecule and hybridizes with it to form a double strand. The free 3'-hydroxyl end of the double strand can serve as a substrate for DNA polymerase and as the starting point of the polymerization reaction that synthesizes the entire single strand into a double strand. Primers are used specifically in PCR, the polymerase chain reaction, which is known to those skilled in the art.

"Plasmid" refers to a nucleic acid molecule that is not integrated into a chromosome but is present in many prokaryotic microorganisms and some eukaryotic microorganisms and is approximately 2 kb to over 200 kb in length.

"Ligation" refers to the connection of the ends of two nucleic acid molecules by ligase or self-ligation. Self-ligation refers to the connection completed by intramolecular ring closure reaction, in which two single-stranded ends of a linear DNA molecule can form bases with each other Yes, the two will dimerize.

"Restriction endonuclease" refers to a class of bacteria-produced enzymes capable of cleaving phosphodiester bonds in specific base sequences of DNA double-stranded molecules.

"Electroporation" is a method of mediating the entry of nucleic acids into cells. Therefore, when the recipient cells are exposed to the nucleic acid solution, the cell membranes of the recipient cells grown in suspension to the logarithmic phase are partially permeabilized with high molecular weight molecules by using short electric pulses of high field strength.

"Overexpression" is understood to mean that the cell produces an increased production of functional plasminogen compared to the production of a wild-type cell. Generally, overexpression means that when produced in a cell, the expression level of the exogenous gene accounts for about 1-40% of the total amount of the total intracellular protein of the host cell.

It should be understood that "expression vector" refers to such a vector that, when the vector enters a suitable host cell, it can transcribe the foreign gene cloned into the vector and then translate it into mRNA (messenger RNA). Expression vectors typically contain control signals necessary for gene expression in prokaryotic or eukaryotic cells.

In the present invention, preferred are methanol-inducible promoters such as the AOX1-promoter, or particularly preferred constitutive promoters such as the YPT1-promoter or GAP-promoter, which are used to control the expression of genes in yeast expression, the yeast such as Pichia pastoris. Particular preference is given to the constitutive GAP-promoter.

"AOX1" is the gene of alcohol oxidase 1 derived from Pichia pastoris;

"GAP" is the gene for glyceraldehyde-3-phosphate dehydrogenase derived from Pichia pastoris; and

"YPT1" is a gene for a GTP-binding protein derived from Pichia pastoris.

Signal peptides of proteins encoded by PHO-1, SUC-2, PHA-E or α-MF genes are usually used in yeast secretion products.

"PH01" is an acid phosphatase gene derived from Pichia pastoris;

"SUC-2" is a secreted invertase gene derived from Saccharomyces cerevisiae;

"PHA-E" is the acid phosphatase gene derived from Phaseolus vulgaris Agglutinis;

"α-MF" is an α-hybridization factor gene derived from Saccharomyces cerevisiae.

Particularly preferred are the codons for the cleavage point of the protease, and the codons for the cleavage point of the propeptide by the protease Kex2 or Ste13. It is particularly preferred to carry out the linking in step a) above using codons which code for the Kex2 cleavage point and the other two Ste13 cleavage points. In a preferred embodiment of the present invention, the nucleic acid molecule encoding the signal peptide or the propeptide is derived from yeast, especially from Saccharomyces cerevisiae. In a more preferred embodiment, it relates to a nucleic acid molecule encoding a signal peptide or a prepropeptide encoding a signal peptide or a prepropeptide of the alpha-factor of Saccharomyces cerevisiae. The fusion product formed as described in step a) above is preferably amplified by PCR and further preferably suitably purified.

In patent application publication number WO02/50290, the recombinant production of small plasminogen and microplasminogen by the expression vector pPICZαA suitable for yeast, which contains the inducible AOX1-promoter and the yeast α - the prepro-pro-peptide of the factor. These smaller plasminogen variants have no (eg microplasminogen), or only one (eg miniplasminogen) ring cake domain. The expression vector pPICZαA contains protease Kex2 and Ste13 cutting sites. However, in the corresponding expression vectors of small plasminogen and microplasminogen, Ste13 cutting point was deleted.

A set of promoters for inducible expression systems in yeast is known. Included so far in particular, AOX1-promoter, AOX2, CUP1 (Koller A, Valesco J, Subramani S., Yeast 2000: 16(7), 651-6), PH01 (EP0495208), HIS4 (US 4885242), FLD1 ( Shen et al., Gene 1998:216(1), 93-10) and the XYL1-promoter (Den Haan and Van Zyl, Appl. Microbiol. Biotechnol. 2001:57(4), 521-7).

Through the method of methanol-induced AOX1-promoter, heterologous proteins can be selectively and directionally produced, and uniform quantities can be obtained. The host organism is allowed to achieve high growth densities without selection defects that occur in expressing the foreign protein prior to inducing the foreign protein for expression.

In the patent application with the publication number WO02/50290, a smaller variant was expressed under the control of the AOX1-promoter, contrary to the smaller variant, the recombinantly produced plasminogen glutamate and Lysine plasminogen contains all five ring cake domains, complicating its recombinant production for the following reasons:

-When the host expresses the foreign protein, due to its growth defect, the expression element may be lost;

- proteolytic degradation of the expressed protein; and

— Low yield.

Due to said drawbacks, the production of glutamate plasminogen and lysine plasminogen is not disclosed in patent application publication number WO 02/50290.

In the present invention these difficulties have been solved in particular by the recombinant protein comprising a signal peptide, Kex2 and at least one Ste13, preferably two Ste13 protease cleavage sites. Further, in a preferred embodiment, glycerol as another carbon source is supplied at a rate of 0.1 to 10 ml/h, more preferably 0.5 to 5 ml/h, further preferably 0.8 to 1.5 ml/h. The medium is a neutral buffer system with pH 7.0. Pay attention to ensure adequate oxygen supply.

In a preferred embodiment, it should be noted that the recombinant nucleic acid is not connected to the 5' end of the AOX1 gene, but to the 5' end of the phosphate glyceraldehyde dehydrogenase gene derived from Pichia pastoris. Here, a constitutive, non-inducible promoter was used. Constitutive promoters active in yeast that can be used are the GAP-promoter, the YPT1-promoter (Sears et al., Yeast 1998: 14(8), 783-90), the TKL-promoter (Den Haan and Van Zyl, Appl.Microbiol.Biotechnol.2001: 57(4), 521-7), ACT-promoter (Kang et al., Appl.Microbiol.Biotechnol.2001: 55(6), 734-4" and PMA1-promoter (Yeast2000 : 16(13), 1191-203). Preferred promoters are the GAP-promoter and YPT1-promoter. Particularly preferred promoters are the GAP-promoter.

In contrast to inducible promoters, constitutive promoters have the drawback that the foreign protein to be expressed is continuously produced constitutively throughout the growth cycle. This is an unfavorable condition for the host cell, especially manifested by retarded growth of the host cell. Host cells that have lost the recombinant expression elements have a growth advantage due to dominant selection pressure and can overgrow the recombinant host cells. Thus, heterogeneous mixed populations can occur, which should be avoided. It has however been surprisingly found that, according to a preferred embodiment of the present invention, a constitutive GAP-promoter leads to a higher yield.

When the AOX1-promoter is used, the production of lysine plasminogen can be obtained at least 17 U/l (unit/liter) (=1.5 mg/l) after induction for 120 hours, more preferably 120 U/l (=11 mg /l), more preferably 180U/l (=16mg/l), more preferably 200U/l (=18mg/l), more preferably 220U/l (=20mg/l), and even more preferably 240U/l l (=22 mg/l), particularly preferably 260 U/l (=24 mg/l), more particularly preferably 280 U/l (=25.5 mg/l); when using a constitutive promoter, especially the GAP-promoter , the yield increased significantly.

In a preferred embodiment, a constitutive promoter such as the GAP-promoter is operably coupled to a nucleic acid encoding at least a functional portion of the plasminogen sequence and to a nucleic acid sequence encoding at least one signal peptide Fusion, the nucleic acid sequence encoding the functional plasminogen and the nucleic acid sequence encoding at least the signal peptide are coupled with a protease cleavage site codon, and the protease is used to cut the signal peptide.

In a particularly preferred embodiment, a constitutive promoter, such as the GAP-promoter, is operably coupled to control a micro-, small-, Lys- or Glu-plasminogen nucleic acid sequence, which is identical to the yeast-derived Nucleic acid sequence fusion of signal peptide.

This can be seen as a surprising finding that, according to a preferred embodiment of the invention, a constitutive GAP-promoter can achieve a 10-fold higher yield (see Example 7c, Production of lysine plasminogen, 1375U/l, which can be converted to 125mg/l). In another preferred embodiment, glycerol is supplied as another carbon source at a rate of 0.1 to 10 ml/h (milliliters per hour), more preferably of 0.5 to 5 ml/h, still more preferably of 0.8 to 1.5 ml/h, The medium is a neutral buffer system with pH=7.0. Accordingly, the growth rate μ [l/h] is between 0.002 and 0.10, preferably between 0.004 and 0.020, more preferably between 0.008 and 0.010.

In the application of the GAP-promoter, a yield of lysine plasminogen of at least 660 U/l (= 60 mg/l), preferably 1000 U/l (= 91 mg/l) is obtained after the fermentation lasts 250 hours, More preferably 1500U/l (=136mg/l), more preferably 2000U/l (=182mg/ml), particularly preferably 2500U/l (=227mg/l), further particularly preferably 2750U/l (=250mg/ml) l).

Therefore, recombinant small plasminogen and microplasminogen products can obtain higher expression levels. The production of small plasminogen is between 100 mg and 2 g per liter, preferably 300 mg/l-1.5 g/l, more preferably 400 mg/l-1 g/l, still more preferably 500 mg/l-800 mg/l , particularly preferably 600 mg/l-700 mg/l. The production of microplasminogen is at least 10% higher than that of miniplasminogen. Compared with the lysine plasminogen recombinant product, the yield of the obtained glutamate plasminogen recombinant product was slightly lower.

The method of the present invention is suitable for producing small-, micro-, Lys- and Glu-plasminogen. Therefore, preferred embodiments also focus on the recombinant production of small-, micro-, Lys- and Glu-plasminogen, which are all combined with Signal sequence or prepro sequence (prepro sequence) coupling, these vectors contain constitutive promoters such as GAP-promoter. In a further preferred embodiment, the signal sequence consists of a signal peptide or the propeptide of factor a of Saccharomyces cerevisiae. In particularly preferred embodiments, a constitutive promoter, such as the GAP-promoter, is operably associated with one of Seq.ID.No.7 or 9, or Seq.ID.No.13 or 15, or Seq.ID.No.13 or 15, or Seq.ID.No. . No. 50 to 59 in a nucleic acid sequence coupled, and expressed in the appropriate expression vector.

In another preferred embodiment, a constitutive promoter, such as the GAP-promoter, is operably coupled to a nucleic acid sequence encoding at least a functional part of plasminogen. In a particularly preferred embodiment, a constitutive promoter, such as the GAP-promoter, is operably associated with Seq.ID.No.13, 15, 7 and 9, or Seq.ID.No.50 to 59 One is coupled, or coupled with the nucleic acid sequence of Seq.ID.No.11, and expressed in an appropriate expression vector.

Plasminogen glutamate (data calculated with EditSeq ^TM (DNASTAR) program)

Molecular weight: 88431.67Dalton (Dalton)

791 amino acids

When pH=7.0, the isoelectric point of 7.121 is 1.351

Glycosylation sites: 0-268, N-308, 0-365

(Numbering according to the sequence of preplasminogen consisting of 810 amino acids)

Lysine plasminogen (data calculated with EditSeq ^TM (DNASTAR) program)

Molecular weight: 79655.71Dalton

741 amino acids

When pH=7.0, the isoelectric point of 7.492 is 5.287

Glycosylation sites: 0-268, N-308, 0-365

(Numbering according to the sequence of preplasminogen consisting of 810 amino acids)

Small plasminogen (data calculated with EditSeq ^TM (DNASTAR) program)

Molecular weight: 38169.63Dalton

348 amino acids

When pH=7.0, the isoelectric point of 7.203 is 0.893

Glycosylation site: no

Microplasminogen (data calculated with EditSeq ^TM (DNASTAR) program)

Molecular weight: 27230.41Dalton

249 amino acids

When pH=7.0, the isoelectric point of 7.934 is 3.733

Glycosylation site: no

The method of the present invention is described in detail as follows:

The fusion product produced in step a) of the present invention can be integrated into an expression vector suitable for microorganisms. The expression vector is preferably selected from the following vectors: pPICZαA, B and C, pPICZA, B and C, pGAPZαA, B and C, pGAPZA, B and C, pPIC6αA, B and C, pPIC6A, B and C, and pAO815, pPIC3.5K and pPIC9K. More preferably, the method of ligation is used to introduce the expression vector. The PCR product and expression vector are preferably digested with restriction endonucleases KspI and XhoI prior to ligation with T4 DNA ligase. The ligated nucleic acid can be transformed into microorganisms such as Escherichia coli by electroporation, and DNA can be extracted from the obtained microorganisms, and preferably digested and separated using endonucleases XhoI or SfuI and KspI. The nucleic acid thus obtained may be a plasmid, preferably selected from the group consisting of pMHS476.1, pSM54.2, pSM49.8, pSM82.1, und pSM58.1, pAC37.1, pJW9.1, pPLG1.1, pPLG2.1, pPLG3.2, pPLG4.2, pPLG5.3, pPLG6.1, pPLG7.1, pPLG8.3, pPLG9.1, pPLG10.1, pPLG11.2, pPLG11.2, pPLG13.1, pPLG14. 2. pPLG15.1, pPLG16.3, pPLG17.2, pPLG18.1, pPLG19.2 or pPLG20.1. The two oligonucleotide primers used for the above-mentioned amplification can preferably be selected from the following sequences: NO34 (Seq.ID No.1), NO36 (Seq.ID No.2), NO36a (Seq.ID No.19), NO36b (Seq.ID No.20), NO36c(Seq.ID No.21), NO36d(Seq.ID No.22), NO36e(Seq.ID No.23), NO36f(Seq.ID No.24), NO36g (Seq.ID No.25), NO36h(Seq.ID No.26), NO36i(Seq.ID No.27), NO36j(Seq.ID No.28), NO57(Seq.ID No.3), NO37( Seq.ID No.4), NO35(Seq.ID No.5) or NO56(Seq.ID No.6).

According to the invention, the following embodiments are particularly preferred:

-The codon of the protease Kex2 restriction site and plasminogen fusion gene, its characteristic nucleic acid sequence is shown in the sequence of Seq.ID No.7 or 13.

-Codons encoding protease Kex2 restriction site and plasminogen fusion protein, its characteristic amino acid sequence features are shown in the sequence of Seq.ID No.8 or 14.

-Codons of protease Kex2, Ste13 restriction site and plasminogen fusion gene, its characteristic nucleic acid sequence is shown in the sequence of Seq.ID No.9 or 15.

-Codons encoding protease Kex2, Ste13 restriction site and plasminogen fusion gene, its characteristic amino acid sequence is shown in the sequence of Seq.ID No.10 or 16.

Preferably, the above-mentioned plasmid selected from the above-mentioned plurality of plasmids is transformed into a host microorganism. Transformation methods such as electroporation may be employed. The microorganisms used are preferably eukaryotic microorganisms, which are a branch of fungi. Preferred microorganisms are Ascomycota, among which Sacchariomycotina is preferred, Saccharomycetes is more preferred, Saccharomycetales is still more preferred, Saccharomycetaceae is still more preferred, Pichia, Saccharomyces, Hansenula and Aspergillus are particularly preferred Genus (Aspergillus).

According to a particularly preferred embodiment of the present invention, in the host microorganism transformed with the fusion product produced in step a), the nucleic acid sequence encoding at least the functional part of plasminogen is overexpressed, and at least the plasminogen is secreted The profunctional part is preferably secreted into the culture medium. According to another preferred embodiment, the nucleic acid sequence of the functional part of plasminogen is one of the sequences Seq. ID No. 60, 61, 62, 63, 64, 65 or 66. According to another preferred embodiment, the nucleic acid sequence of the functional part of plasminogen is identical to the complete sequence of plasminogen. Using the recombinant product production method of the present invention, it is preferred to produce human functional plasminogen.

The plasminogen obtained according to the production method of the recombinant product described in the present invention, or the plasminase obtained after the plasminogen is acted on by protease, can be used to produce medicines for treating wounds, especially for treating slow healing. Or non-healing wounds, can also be used to treat thrombotic events or prevent thrombotic events.

Furthermore, it was found that the plasminogen produced according to the invention and the plasmin obtained therefrom have anticoagulant properties. These advantageous properties make plasminogen and/or plasmin, also useful as antithrombotic and anticoagulant agents, useful for the prevention and/or treatment of the following diseases: heart attack, thrombosis, heart valve revascularization Stenosis, Hypoxia, Ischemia, Coagulative Necrosis, Vasculitis; May be used in subsequent treatment of: Heart Attack, Bypass Surgery, Angioplasty, Balloon Dilatation. Plasminogen is also used in thrombolytic therapy in acute heart attack, recanalization of arteriovenous short circuit and reperfusion of occluded coronary arteries in acute heart attack. The plasminogen produced according to the present invention can also be used to prevent and treat the following diseases: acute pulmonary embolism, new or old venous thrombosis, acute and subacute arterial thrombosis, venous thrombosis, acute extremity arterial obstruction , chronic occlusive arterial disease, thrombosis in arteriovenous shorts, buttock and extremity deep vein thrombosis, early thrombosis in areas of desobliterated vessels, acute ocular central vessel embolism, conjunctivitis due to type I plasminogen deficiency , burns and frostbite, alkali or acid burns, and disseminated intravascular coagulation in shock.

When suffering from the above-mentioned diseases, it is preferable to use plasminogen and/or plasmin together with anticoagulants. Suitable anticoagulants are heparin, heparin derivatives or acetylsalicylic acid.

Therefore, the pharmaceutical composition of the present invention is also very important, and the pharmaceutical composition comprises: the plasminogen obtained according to the recombinant product production method of the present invention or the plasmin obtained therefrom, and the required pharmacological Acceptable substrates, additives and/or solvents. Furthermore, it is preferred that the pharmaceutical composition may comprise an anticoagulant active agent, especially heparin, a heparin derivative or acetylsalicylic acid.

The plasminogen produced according to the invention and/or the plasmin obtained therefrom are used in the in vitro treatment of wounds, preferably in the form of a pharmaceutical composition for local treatment of wounds. In this application, the concentration of plasminogen and/or plasmin is 0.01-500U of plasminogen and/or plasmin per gram of the pharmaceutical composition, preferably 0.1-500U, more preferably 0.1-250U, more preferably 0.5-250U, particularly preferably 1-150U. If plasters or other dressings are used instead of semi-solid formulations such as ointments, pastes or gels, the above given drug concentration and dressing surface area per ² cm2 of plaster surface area should be considered separately.

The pharmaceutical composition of the present invention is produced according to known methods and appropriate doses for the desired type of application, using commonly used solid or liquid bases or diluents and commonly used pharmaceutical adjuvants. The preferred pharmaceutical preparation or composition is a pharmaceutical dosage form suitable for topical application. The pharmaceutical dosage form is, for example, ointment, paste, gel, coating, powder, emulsion, suspension or other special formulations, such as nano-dispersion systems, including liposomes, nanoemulsions or lipid nanoparticles. forms, including surfactant-free formulations, polymer-stabilized or particle-stabilized emulsions.

Methods for the production of various formulations and different methods of application are known to those of ordinary skill in the art and are described in detail, for example, in "Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton PA".

Formulation of the pharmaceutical composition for parenteral administration is appropriate in the following cases: The pharmaceutical composition is used for the prophylaxis and/or treatment of heart attack, thrombosis, restenosis after heart valve surgery , hypoxia, ischemia, coagulative necrosis, vasculitis, and acute heart attack; and for subsequent treatment of: heart attack, bypass surgery, angioplasty, and balloon dilatation.

In addition, the pharmaceutical composition is also suitable for various systemic applications, including acute pulmonary embolism, thrombolytic therapy in acute heart attack, new or old venous thrombosis, acute and subacute arterial thrombosis, arteriovenous short circuit Recanalization, venous embolism, reperfusion of occluded coronary arteries in acute heart attack, acute extremity arterial occlusion, chronic occlusive arterial disease, arteriovenous short circuit thrombosis, buttock and extremity deep vein thrombosis, early thrombosis in areas of occluded vessels , acute ocular central vascular embolism, conjunctivitis due to type I plasminogen deficiency, burns, alkali or acid burns and frostbite, and disseminated intravascular coagulation during shock.

The pharmaceutical composition can also be used in cases of plasminogen deficiency, such as hereditary or congenital (congenital) plasminogen deficiency (homozygous type I plasminogen deficiency), which can Lead to conjunctivitis lignosa or thrombophilic state. The aforementioned diseases can also be treated, for example, by intravenous injection of recombinant plasminogens, including Glu-, Lys-, small- and micro-plasminogen and their derivatives (Heinz et al., Klin. Monatsblatt Augenheilkunde 2002, 219(3):156-8).

Another possible application is the elimination of airway pseudomembranes, normalization of the airways and faster wound healing through the administration of plasminogen. Said application has been used in neonates (The New England Journal of Medicine 1998, 339, 23, 1679-1686).

Recombinantly produced plasminogen is used together with plasmin thus obtained, or only plasmin is added to the pharmaceutical composition, which is suitable for the prevention and/or treatment of the following diseases: acute pulmonary embolism, new-onset or Old venous thrombosis, acute and subacute arteriovenous thrombosis, arteriovenous short circuit recanalization, venous embolism, reperfusion of occluded coronary arteries in acute heart attack, acute extremity arterial occlusion, chronic occlusive arterial disease, arteriovenous short circuit Thrombosis, buttock and extremity deep vein thrombosis, early thrombosis in areas of occluded vessels, acute ocular central vessel embolism, conjunctivitis due to type I plasminogen deficiency, burns, alkali or acid burns and frostbite, and diffuse in shock Sexual intravascular coagulation.

Preference is given to pharmaceutical compositions containing recombinantly produced plasminogen according to the invention, which may be suitable for topical treatment of burns, frostbite, alkali or acid burns, injuries and/or wounds, especially non-healing wounds. Preferably the recombinant plasminogen is used together with at least one activator, which may be eg urokinase or streptokinase. Another preferred method is, before using the plasminogen produced by the present invention, fully or partially activate plasminogen into plasmin by an activator, and use it for the above-mentioned indications, the pharmaceutical preparation of this method Including, plasmin alone or plasmin plus plasminogen.

Particularly contemplated routes of parenteral administration are intravenous, intraarterial, intraperitoneal, subcutaneous and intramuscular administration. If parenteral administration is adopted, the concentration of protein in the solution for injection or infusion is in the range of 0.1-100 million units/10ml solution, preferably 10-100 million units/10ml, more preferably 1-10 million units/10ml, particularly preferably 3-5 million units/10ml. If it is administered orally, the protein concentration is 0.1-100,000 units/gram, preferably 100-80,000 units/gram, particularly preferably 1,000-50,000 units/gram.

Pharmaceutical preparations are of further advantage, for example plasters, dressings or other topical substances containing proteases. These formulations are particularly suitable for topical application in wound healing, or for the treatment of burns, frostbite, alkali or acid burns and/or injuries. Preferably, the recombinantly produced plasminogen of the present invention is used in pharmaceutical compositions, especially wound healing agents, ointments and other materials for external application, which are combined with at least one activator (the plasminogen Original activators such as urokinase, streptokinase), or the plasmin that is formed by the activation of the aforementioned activators, are used together, and in and/or on the pharmaceutical composition and preparation, plasmin is potentially combined with plasminogen Used together, or potentially with at least one activator. The use of plasminogen, preferably plasminogen and an activator; or plasmin or plasmin plus plasminogen and an activator is particularly preferred in and/or on plasters and topical substances, so The preparation is suitable for wound treatment, especially for the treatment of refractory wounds, and can also be used for the treatment of burns, frostbite, alkali or acid burns, or other injuries.

Dressings, wound healing dressings or plasters, containing plasminogen produced by the present invention and/or plasmin obtained therefrom, wherein the pharmaceutical preparation contains 0.01-500U plasminogen and/or plasmin per square centimeter, Preferably, each square centimeter of the dressing or plaster contains 0.1-500 U of plasminogen and/or plasmin, respectively. Preferably, in the pharmaceutical preparation per square centimeter of the plaster or dressing, the concentration of plasminogen and/or plasmin produced therefrom is 0.1-250U, more preferably 0.5-250U, especially preferably 1-150U .

To activate 1 mg of plasminogen, the dosage of urokinase is 1 ng (nanogram)-100 μg (microgram), preferably 10 ng-10 μg. To activate 1 mg of plasminogen, the dosage of streptokinase is 1 μg-1 mg, preferably 3 μg-300 μg. To activate 1 mg of plasminogen, the amount of protease derived from Streptomyces griseus (S. griseus) is 10 ng-100 μg, preferably 100 ng-10 μg. To activate 1 mg of plasminogen, the amount of protease VIII is 10 ng-100 μg, preferably 100 ng-10 μg.

The nucleic acid sequence encoding a functional part of plasminogen is preferably a DNA sequence.

In addition, the plasmids related to the present invention are as follows:

Plasmid pPLG1.1

Plasmid pPLG2.1

Plasmid pPLG3.2

Plasmid pPLG4.2

Plasmid pPLG5.3

Plasmid pPLG6.1

Plasmid pPLG7.1

Plasmid pPLG8.3

Plasmid pPLG9.1

Plasmid pPLG10.1

Plasmid pPLG11.2

Plasmid pPLG11.2

Plasmid pPLG13.1

Plasmid pPLG14.2

Plasmid pPLG15.1

Plasmid pPLG16.3

Plasmid pPLG17.2

Plasmid pPLG18.1

Plasmid pPLG19.2

Plasmid pPLG20.1

Plasmid pMHS476.1 (Accession No.: DSM 14678)

Plasmid pSM54.2 (Accession No.: DSM 14682)

Plasmid pSM49.8 (Accession No.: DSM 14681)

Plasmid pSM82.1 (Accession No.: DSM 14679)

Plasmid pSM58.1 (Accession No.: DSM 14680)

Plasmid pAC37.1 (Accession No.: DSM 15369)

Plasmid pJW9.1 (deposit number: DSM 15368).

(The deposit number refers to the deposit number of the German Collection of Microorganisms and Cell Cultures Ltd. (GermanCollection of Microorganisms and Cell Cultures Ltd., Mascheroder Weg 1b, D-38124 Braunschweig).

In addition, the present invention relates to a DNA sequence suitable for expression, which comprises a nucleic acid sequence encoding at least a functional part of plasminogen, which can be obtained according to the recombinant production method described in the present invention. Furthermore, the present invention also relates to a host microorganism comprising a fusion product, see step a) above, and a nucleic acid sequence derived therefrom. The invention also relates to vectors, DNA molecules or RNA molecules comprising a fusion product, see step a) above, and a nucleic acid sequence derived therefrom.

Finally, the present invention also relates to a screening method for identifying plasminogen activators, especially plasminogen activating proteases, wherein the functional plasminogen produced according to the production method of the above-mentioned recombinant product is used . For this purpose, plasmin obtained after protease pre-incubation is preferred for measuring activity together with functional plasminogen produced according to the method of the present invention. A synthetic peptide can be used to measure the activity of the resulting plasmin. It is particularly preferred to use N-tosyl (tosyl)-Gly-Pro-Lys-pNA to measure the activity of the resulting plasmin.

Description of drawings

Use accompanying drawing to explain the present invention in more detail, accompanying drawing is as follows:

Figure 1: Physical map of plasmid pMHS476.1 (5682bp). The α-factor propeptide progene is linked with the human lysine plasminogen gene through the codon of the Kex2 restriction site, and the α-factor propeptide progene is under the control of the AOX1 promoter.

Figure 2: Physical map of plasmid pSM54.2 (5694bp). The α-factor propeptide progene is connected with the human lysine plasminogen gene through a codon of Kex2 and two Ste13 restriction sites, and the α-factor propeptide gene is under the control of the AOX1 promoter.

Figure 3: Physical map of plasmid pSM49.8 (5715bp). The human preplasminogen gene is under the control of the AOX1 promoter.

Figure 4: Physical map of plasmid pSM82.1 (5913bp). The α-factor propeptide progene is linked with the human lysine plasminogen gene through the codon of the Kex2 restriction site, and the α-factor propeptide progene is under the control of the AOX1 promoter.

Figure 5: Physical map of plasmid pSM58.1 (5925bp). The α-factor propeptide progene is connected with the human glutamic acid plasminogen gene through a codon of Kex2 and two Ste13 restriction sites, and the α-factor propeptide gene is under the control of the AOX1 promoter.

Figure 6: Physical map of plasmid pAC37.1 (11400bp). The α-factor propeptide progene is connected with the human lysine plasminogen gene through a codon of Kex2 and two Ste13 restriction sites, and the α-factor propeptide gene is under the control of the AOX1 promoter.

Figure 7: Physical map of plasmid pJW9.1 (5925bp). The α-factor propeptide gene is connected with the human lysine plasminogen gene through a codon of Kex2 and two Ste13 restriction sites, and the α-factor propeptide gene is under the control of the GAP promoter.

Figure 8: Physical map of plasmid pPLG1.1. The α-factor propeptide progene is linked with the human small plasminogen gene through a codon of Kex2 and two Ste13 restriction sites, and the α-factor propeptide progene is under the control of the AOX1 promoter.

Figure 9: Physical map of plasmid pPLG11.2. The α-factor propeptide gene is connected with the human small plasminogen gene through a codon of Kex2 and two Ste13 restriction sites, and the α-factor propeptide gene is under the control of the GAP promoter.

Figure 10: Detection of fibrinolytic activity by Klörhof assay.

Fig. 11: Results of 7.5% SDS-PAGE electrophoresis of the fraction purified in Example 6g.

Detailed ways

According to the invention, all microorganisms capable of possessing the properties of glycosylation and, if desired, secretion of proteins can be used as host organisms. Examples include: Saccharomyces cerevisiae, Pichia pastoris, Pichia methanolica, filamentous fungi of the genus Aspergillus polymorpha.

The use of functional plasminogen and plasmin produced separately according to the invention is particularly envisaged in pharmaceutical preparations. In such formulations, functional plasminogen can be mixed with pharmaceutically acceptable bases, adjuvants and other adjuvants or additives known to those skilled in the art.

There is a Kex2 cleavage site on the propeptide which can be cleaved by Kex2 protease located in the Golgi apparatus. This protease, also known as protease YscF or Kexin, is a serine protease capable of processing proproteins by cleaving the C-terminus from basic amino acid pairs (such as Lys-Arg).

There is a Ste13 cleavage site on the propeptide which can be cleaved by Ste13 protease located in the Golgi apparatus. Protease Ste13 (also known as protease YscVI or dipeptide aminopeptidase A) is distributed in the late Golgi apparatus and can gradually hydrolyze the N-terminal Xaa-Ala dipeptide, such as the N-terminal Xaa-Ala di peptide.

In addition to the cleavage sites for the proteases Kex2 and Ste13, other cleavage sites can also be inserted which are recognized as substrates by proteases located in the endoplasmic reticulum or the Golgi apparatus.

It is also possible to fuse only to the signal sequence (propeptide) of the plasminogen gene, which is responsible for the transport of the product into the endoplasmic reticulum, ie hybridization factor propeptides such as Saccharomyces cerevisiae are not necessary in the process.

Those skilled in the art are familiar with the methods of microbiology, molecular biology and protein chemistry referred to in the examples. The following is a bibliography: Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor press, 1989(10); Gassen & Schrimpf, Genetic Engineering Methods , Spektrum Akademischer Verlag, Heidelberg, 1999 (11); EasySelect ^™ Pichia Expression Kit Instruction Manual (EasySelect ^™ Pichia Expression Kit Operation Manual), Invitrogen, Groningen, Netherlands, catalog number: K1740-01. Both Pichia pastoris strains and expression systems are commercially available from Invitrogen and are described in the above manual.

pPICZA, B and C are 3.3 kb Pichia pastoris expression vectors, which are relatively short. There is a zeocin resistance gene on the vector, which is used to directly select Pichia pastoris transformants. In addition, there is a C-terminal tag sequence on the vector for rapid purification and detection of fusion proteins. pPICZalpha A, B, and C are 3.6kb Pichia pastoris expression vectors, which also have the Meiling resistance gene and the above-mentioned C-terminal marker sequence. In addition, they contain the S. cerevisiae alpha factor secretion signal for efficient protein secretion into the medium.

In addition, plasminogen can be activated. Plasminogen can be activated by incubation with a protease identified using the screening method of the present invention.

Preferably, plasminogen is incubated with the following enzymes: protease from Streptomyces griseus, protease VIII, protease XVIII, ficin, metalloendopeptidase, clostripain, glutamate-C-terminal endoprotease, protease XIII, Protease A, trypsin, aspartate-N-terminal endoprotease or elastase.

It is further conceivable to activate plasminogen by incubation of t-PA, u-PA or vb-PA [vampire bat-PA] with plasminogen.

In another preferred embodiment, plasminogen is activated by incubation with staphylokinase and streptokinase. Streptokinase or staphylokinase forms a 1:1 complex with plasminogen. Through the formation of this complex, the conformation of plasminogen in the complex changes so that it has proteolytic activity and can activate plasminogen to convert it into plasmin.

The functional plasminogen obtained according to the recombinant method, or the activated functional plasminogen, has the ability to hydrolyze fibrin. The plasminogen also has the ability to activate pro-matrix metalloproteinases and growth factors.

The present invention will be illustrated in more detail by the following examples.

Example 1a: Amplification of the lysine plasminogen gene, the gene is inserted into the codon of the Kex2 restriction site at the 5' end

Using the QIAGEN plasmid extraction kit (QIAGEN, Hilden), the plasmid pPLGKG was extracted from the HB101 Escherichia coli strain (containing pPLGKG) (Forsgren et al., FEBS Lett. March 23, 1987; 213(2):254-60(2 )), which contains the pre-glutamate-plasminogen (pre-Glu-plasminogen) gene. 150 ng of pPLGKG-DNA was digested with 10 U of EcoRI restriction enzyme (Roche, Mannheim) to linearize it, and then the DNA was purified using the QIAquick PCR purification kit (QIAGEN, Hilden). The plasminogen gene was amplified with primers NO34 (Seq.ID No.1) and NO36 (Seq.ID No.2). In addition to the base complementary to the plasminogen gene, the oligonucleotide primer NO36 also has the codon of the Kex2 restriction site. Each reaction system contained 0.5U Pwo-DNA-polymerase (Hybaid, Heidelberg), 400 nM oligonucleotide primers, 200 μM dNTPs, 3 ng linear pPLGKG-DNA and respective reaction buffers in a final volume of 50 μl. The primer binding temperature was 58°C.

Check whether the expected size of the PCR product band is correct by agar gel electrophoresis, and purify it with the QIAquick PCR purification kit.

Example 1b: Cloning of the plasminogen gene into the pPICZaA vector

400 ng of the PCR product was digested with 10 U each of KspI and XhoI (Roche, Mannheim) restriction enzymes. Plasmid pPICZαAA (Invitrogen, Groningen, Netherlands) contains the pre-propeptide sequence derived from Saccharomyces cerevisiae α-factor, and 300 ng of the DNA of the plasmid was digested with 10 U of KspI and XhoI restriction endonucleases. The digested DNA was separated on 0.9% agarose gel, and the obtained fragments were recovered from the gel with a QIAquick gel extraction kit (QIAGEN, Hilden). The vector DNA and insert DNA were mixed and ligated overnight at 4°C with 1 U T4-DNA-ligase (Roche, Mannheim).

Subsequently, the QIAquick PCR purification kit was used to purify the batch of ligation product DNA, and the purified product was used to transform Escherichia coli JM109 by electroporation.

The JM109 Escherichia coli cells transformed by electroporation were inoculated into 1ml of SOC medium, cultured at 37°C for 1 hour, and then spread on LB agar solid agar base with 20 μg/μl Meiyilin (Invitrogen, Groningen, Netherlands) , and incubated overnight at 37°C.

From one of the Escherichia coli thus obtained, DNA was extracted using the QIAGEN plasmid mini kit (QIAGEN, Hilden), digested with XhoI and KspI restriction enzymes, and separated by agarose gel electrophoresis Out 300ng nucleic acid. The isolated plasmid contained a fragment of the expected size and was designated pMHS476.1 (Figure 1). Through sequence analysis, it was determined that the sequence of the fusion gene was correct, and the fusion gene was composed of codons derived from the Saccharomyces cerevisiae α-factor propeptide progene, lysine plasminogen gene and protease Kex2 restriction site (Seq. ID No. 7).

Example 1c: Transformation of Pichia pastoris with plasmid pMHS476.1

Using QIAGEN plasmid midi kit (QIAGEN plasmid midi kit, QIAGEN, Hilden) from JM109 Escherichia coli strain (containing pMHS476.1), the expression vector pMHS476.1 plasmid DNA of plasminogen was extracted. Digest 10 μg pMHS476.1-DNA with 100U PmeI (New England Biolabs, Frankfurt) to linearize it, and transform it into Pichia pastoris KM71H strain by electroporation according to the method indicated in the EasySelect ^TM Pichia Expression Kit Operation Manual Among them, the genotype of the KM71H strain is his4::HIS 4 arg 4 aoxl::ARG 4 of Pichia pastoris Y-11430 strain (Northern Regional Research Laboratories, Peoria, USA). After the clones were grown in the YPDS solid medium for three to four days, they were plated and cultured in the YPDS solid medium containing 100 μg/ml Meiyilin, and used to inoculate the liquid medium. The clones were named Pichia pastoris KM71H/pMHS476.1-1/a, where "a" represents the sequential numbering of the clones starting from 1.

Example 1d: Growth of Pichia pastoris KM71H/pMHS476.1-1/1 to -1/3 and induction of plasminogen gene expression

To prepare precultures (precultures), 100ml of BMGY medium (EasySelect ^TM Pichia expression kit operating instructions) was placed in a 1L Schikane flask and cultured at 28°C and 250rpm (rotation per minute) until the OD ₆₀₀ reached 20-30. Then, the preculture was centrifuged at 4645g (centrifugal force) for 10 minutes at 4°C. In order to make the wet mass concentration of the bacterial cells reach 80 g/L, the collected bacteria were resuspended in BMMY-medium (containing 0.5% methanol). 60ml of the main culture was grown in a 300ml Schikane flask for 118 hours at 28°C and 250rpm. 2% methanol was added after 24 and 72 hours. The Schikane flask must be shaken at a high speed of 250rpm to provide sufficient oxygen to the bacterial solution, which is a necessary condition when using the AOX promoter.

Example 1e: Detection of plasminogen activity in primary culture supernatants following activation with streptokinase

A sample of the main culture was centrifuged at 16,000g for 10 minutes. Take 300 μl of supernatant, add 1 μl of streptokinase (S8026) (Sigma, Deisenhofen), and incubate at 37° C. for 20 min. Add 100 μl of N-tosyl-Gly- _Pro -Lys-pNA solution (9.5 mg dissolved in 75mg glycine/10ml, 2% Tween ( ^Tween® ) 20), incubate at 37°C for 10 minutes. The reaction was started by adding 250 μl of supernatant pretreated with streptokinase and further incubated at 37°C. Absorbance was measured at 405 nm, indicating increasing depletion. Parallel groups of Pichia pastoris KM71H culture supernatants as controls, and supernatants not activated with streptokinase were also measured. For example, after the sample has been induced for 72 hours, its activity value (1 U/l is defined as the conversion of 1 μmol N-tosyl-Gly-Pro-Lys-pNA within 1 minute of 1 liter of culture supernatant) is, KM71H/pMHS476.1 -1/1: 2 U/l, KM71H/pMHS476.1-1/2: 2 U/l, KM71H/pMHS476.1-1/3: 1 U/l. After 118 hours of induction, the detected activity values were: 8U/l.

Embodiment 2a: amplify the lysine plasminogen gene, the 5' end of the gene is inserted into a codon of Kex2 and two Ste13 restriction sites

Use two oligonucleotide primers NO34 and NO57 (Seq.ID No.3) to amplify the lysine plasminogen gene, the gene inserts a codon encoding a Kex2 and two Ste13 restriction sites, The amplified product was used to insert into the vector pPICZαA, and the amplification conditions were as described in Example 1a. The oligonucleotide primer NO57 has codons for Kex2 and Ste13 restriction sites next to the complementary bases of the plasminogen gene.

Example 2b: Cloning of the lysine plasminogen gene amplified in Example 2a into the pPICZαA vector

Using a method similar to that of Example 1b, the human lysine plasminogen gene was cloned into the vector pPICZαA to produce a fusion gene consisting of the propeptide progene of Saccharomyces cerevisiae α factor and the inserted proteases Kex2 and Ste13 Human plasminogen gene composition of cleavage site codons. The obtained plasmid was named pSM54.2 (Fig. 2). After sequence analysis, it was confirmed that the sequence (Seq.ID No.9) was correct.

Example 2c: Transformation of Pichia pastoris with plasmid pSM54.2

Pichia pastoris KM71H strain was transformed with plasmid pSM54.2 as described in Example 1c for transformation of pMHS476.1. The obtained clone was named Pichia pastoris KM71H/pSM54.2-1/a, where "a" still represents the serial numbering of the clone starting from 1.

Example 2d: Cultivation of Pichia pastoris KM71H/pSM54.2-1/1 to -1/3 and induction of plasminogen gene

The production of the pre-culture and the main culture, as well as the method of induction with methanol, were similar to those in Example 1d.

Example 2e: Determination of plasminogen activity in samples of the main culture after activation using streptokinase

For the determination of the activity of plasminogen after streptokinase activation, see the description of KM71H/pMHS476.1-1/1 to -1/3 in Example 1e. After 72 hours of induction, the activity values of the samples are as follows: KM71H/pSM54.2-1/1: 2U/l, KM71H/pSM54.2-1/2: 8U/l, KM71H/pSM54.2-1/3: 6U /l. The activity values measured after 118 hours of induction are as follows: KM71H/pSM54.2-1/1: 8U/l, KM71H/pSM54.2-1/2: 17U/l, KM71H/pSM54.2-1/3: 13U /l.

Example 3a: Amplification of the plasminogen gene with its own signal sequence and cloning into the pPICZA vector; transformation of Pichia pastoris

Use two oligonucleotide primers NO34 and NO37 (Seq.ID No.4) to amplify the plasminogen gene, which has a sequence encoding its own signal peptide (preplasminogen), and the amplified product The vector pPICZA was inserted, and the conditions used were as described in Example 1a.

Using a method similar to that described in Example 1b, both the vector and the PCR product were digested with SfuI and KspI restriction enzymes, and then the plasminogen gene was cloned into the pPICZA vector. The obtained plasmid was named pSM49.8 (Fig. 3). After sequence analysis, it was confirmed that the sequence (Seq.ID No.11) was correct.

Pichia pastoris KM71H strain was transformed with plasmid pSM49.8 similarly as described for pMHS476.1 in Example 1c. The resulting clone was named Pichia pastoris KM71H/pSM49.8-1/a, where "a" still represents the serial numbering of the clone starting from 1.

Example 4a: Amplify the human glutamate plasminogen gene and clone it into the expression vector pPICZαA, the human glutamate plasminogen gene is inserted into the codon of the Kex2 restriction site; transform Pasteur Pichia

Using two oligonucleotide primers NO34 and NO35 (Seq.ID No.5), the glutamate plasminogen gene was amplified, and cloned into the vector pPICZαA, the glutamate plasminogen gene was inserted into The codon of the Kex2 restriction site, the conditions used in the process are as described in Example 1a. The oligonucleotide primer NO35 has the codon of the Kex2 restriction site next to the complementary sequence of the glutamic acid plasminogen gene.

The plasminogen glutamate gene was cloned into the vector pPICZαA in order to produce a fusion gene consisting of the propeptide progene of S. Acid plasminogen gene composition, the process is similar to the cloning process described in Example 1b.

The obtained plasmid was named pSM82.1 (Fig. 4). After sequence analysis, it was confirmed that the sequence (Seq. ID No. 13) was correct.

Pichia pastoris KM71H strain was transformed with plasmid pSM82.1 similarly as described for pMHS476.1 in Example 1c. The resulting clone was named Pichia pastoris KM71H/pSM82.1/a, where "a" still represents the serial numbering of the clone starting from 1.

Embodiment 5a: amplify the gene of human glutamate plasminogen and clone into expression vector pPICZa A, the gene of described human glutamate plasminogen inserts coding Kex2 and two Ste13 restriction sites at the 5' end dot codons; transformed Pichia pastoris

Utilize two oligonucleotide primers NO34 and NO56 (Seq.ID No.6), amplify the gene of glutamate plasminogen, and clone in the expression vector pPICZa A, described glutamate plasminogen The codons encoding Kex2 and two Ste13 restriction sites were inserted into the gene of , and the specific amplification conditions are shown in Example 1a. Primer NO56 has codons for Kex2 and Ste13 restriction sites next to the complementary bases of the glutamic acid plasminogen gene.

The gene of human glutamic acid plasminogen was cloned into the vector pPICZa A to produce a fusion gene consisting of the pro-propeptide gene encoding factor a of S. The human glutamate plasminogen gene composition of the dot sequence, the specific steps are similar to those mentioned in Example 1b. The obtained plasmid was called pSM58.1 (Fig. 5). Through sequence analysis, it was confirmed that the sequence (Seq.ID No.15) was correct.

Like the pMHS476.1 described in Example 1c, the Pichia pastoris KM71H strain was transformed with the plasmid pSM58.1, and the resulting clone was named Pichia pastoris KM71H/pSM58.1/a, where " a" still represents the sequential numbering of the clones starting from 1.

Example 6a: Human lysine plasminogen gene was obtained from plasmid pSM54.2 and inserted into vector pPIC9K

150 ng of vector pPIC9K (Invitrogen, Groningen, The Netherlands) was cut with 10 U of each restriction enzyme Sad and Notl (both from Roche Diagnostics, Mannheim). 300 ng of the plasminogen expression vector pSM54.2 was also cut with the same enzyme (see Example 2b). Then the digested DNA fragments were separated by electrophoresis using 0.9% agarose gel. Larger fragments were purified using the QIAgen gel extraction kit (QIAgen gel extraction kit, Qiagen, Hilden). The two larger fragments were mixed and ligated overnight at 4°C with 1 U of T4 DNA ligase.

Escherichia coli DH5α was transformed using a method similar to that described in Example 1b, and the resulting plasmid was purified and identified. However, for the antibiotic for screening transformants, ampicillin was used instead of meiyilin. The plasmid thus constructed was named pAC37.1 (Fig. 6).

Example 6b: Transformation of Pichia pastoris with plasmid pAC37.1

As described in Example 1c for transformation of Pichia pastoris KM71H strain with pMHS476.1, Pichia pastoris KM71 was transformed with plasmid pAC37.1 which had been digested with SalI to render linearization. The transformed cells were inoculated into histidine-free MD-agarose medium (see the multi-copy pichia expression kit instruction manual) and cultivated. The resulting clone was named Pasteur Red yeast KM71/pAC37.1-3/a, where "a" still represents the sequential numbering of the clone starting from 1.

Example 6c: Cultivation of Pichia pastoris KM71/pAC37.1-3/1 and induction of plasminogen gene

The production of pre-culture and main culture and the method of methanol induction were similar to the conditions described in Example 1d. The induction duration was over 216 hours. Initially induced with 0.5% methanol, 24 hours later changed to 2% methanol, every 48 hours and once again 2% methanol.

Example 6d: Determination of Plasminogen Activity in Samples of Primary Cultures Following Streptokinase Activation

The method for measuring the activity of plasminogen after streptokinase activation is consistent with the method for measuring the activity of KM71H/pMHS476.1-1/1 to 1/3 in Example 1e. The activity of the sample taken out after 120 hours of induction was 120U/L; after 216 hours of induction, the measurable activity was 190U/L.

Example 6e: Determination of Plasminogen Activity in Master Culture Samples Following Induction of Pichia pastoris KM71/pAC37.1-3/1 in Minimal Medium (BSM) and Streptokinase Activation

After culturing Pichia pastoris KM71/pAC37.1-3/1 in BMGY complex medium (see Example 1d), 80 g of centrifuged cells were resuspended in 100 ml minimal medium containing minimal salts inducing. Medium Containing Minimal Salts - The minimal medium consists of the following:

H ₃ PO ₄ , 85%: 26.0ml/l; CaCl ₂ 2H ₂ O: 0.6g/l; K ₂ SO ₄ : 18.0g/l; MgSO ₄ 7H ₂ O: 14.0g/l; KOH: 4.0 g/l; glycerin: 20ml/l; antifoaming agent: 1.0ml/l; trace element solution: 8.0ml/l; yeast auxin solution (0.2g/l): 8.0ml/l.

Composition of trace element solution: _H2SO4 : _5.0ml /l; _CuSO4 · _5H2O : 6.0g/l; KI: 0.08g/l; _MnSO4 · _H2O : 3.0g/l; _{Na2MoO 4} : 0.2 g/l; H ₃ BO ₃ : 0.02 g/l; CoCl ₂ : 0.5 g/l; ZnCl ₂ : 20.0 g/l; FeSO ₄ ·7H ₂ O: 65.0 g/l.

For induction, 2% methanol was added daily. After streptokinase is activated, the method for measuring the activity of plasminogen is consistent with the method for measuring the activity after induction of KM71H/pMHS476.1-1/1 to -1/3 in Example 1e. The measured plasminogen activity was 193 U/l after 120 hours of induction and 289 U/l after 168 hours.

Example 6f: Determination of plasminogen activity after streptokinase activation in samples of the main culture in the Klörhof fibrinolysis assay

Klrhof fibrinolysis test [Stack, M.S., Pizzo, S.V., and Gonzalez-Gronow, M. (1992): Effect of desilylation on the biological properties of human plasminogen. (Desialic acid on the biological properties of human plasminogen The impact of), Biochem.J.284,81-86 (13)] was prepared as follows: 1.5 grams of GTG low-melting point agarose was dissolved by boiling into 75 milliliters of 50 mM sodium phosphate buffer at pH=7.4; 35 ml Fibrinogen solution (dissolve 225 mg of fibrinogen per 37.5 ml of 50 mM sodium phosphate buffer with pH = 7.4) was mixed with 350 μl thrombin solution (10 U/ml, prepared with 50 mM sodium phosphate buffer with pH = 7.4) , do not generate air bubbles; stir the mixed solution into the agarose solution, and then pour it into a petri dish. After the fibrin has solidified, a small hole of 1 mm in size is carved on the medium.

To measure the fibrinolytic activity of recombinant plasminogen after streptokinase activation, 20 μl of a solution with the following composition was injected into each well and incubated at 37°C for 20 hours:

1: 0.5 mg/ml plasminogen (Roche, Mannheim);

2: the culture supernatant of KM71/pAC37.1-3/1 in Example 6e;

3: 0.5mg/ml plasminogen, activated by streptokinase;

4: the culture supernatant of KM71/pAC37.1-3/1 in embodiment 6e activated by streptokinase;

5: 0.25mg/ml plasminogen, activated by streptokinase;

6: the culture supernatant of KM71/pAC37.1-3/1 diluted one-fold in Example 6e, activated by streptokinase;

7: 0.125mg/ml plasminogen, activated by streptokinase;

8: KM71H culture supernatant obtained by the same method as KM71/pAC37.1-3/1 in Example 6e, activated by streptokinase.

When streptokinase was activated, 2 μl of streptokinase (100 U/μl, Sigma, Deisenhofen) was added to 40 μl of each solution, and incubated at 37° C. for 60 minutes.

Plaques produced by fibrinolytic activity are shown in Figure 10.

Example 6g: Purification of plasminogen produced recombinantly in Pichia pastoris KM71/pAC37.1-3/1 by affinity chromatography

50 ml of the supernatant of Pichia pastoris KM71/pAC37.1-3/1 from example 6c/6d were dialyzed against 4 liters of 50 mM sodium phosphate buffer at pH=7.5 at 4°C. After 24 hours, the dialysis buffer was changed and dialysis was continued for 24 hours. The dialyzed product was filtered with a filter with a pore size of 0.02 μm, and then affinity was performed on a Lysine-SepharoseTM 4B column (diameter 16 mm, height 95 mm) (Amersham Biosciences) equilibrated with 50 mM, pH 7.5 sodium phosphate buffer. chromatography. Non-specifically bound proteins on the column can be removed by 50 mM sodium phosphate buffer, pH=7.5, 0.5 M sodium chloride. Bound plasminogen can be eluted with 50 mM sodium phosphate buffer, pH 7.5, 0.01 M ε-aminocaproic acid. The obtained samples can be electrophoresed by 7.5% SDS-PAGE, and then stained by silver staining ( FIG. 11 ). On the gel, the recombinant plasminogen contained in the fractions was in the same position as the human plasminogen added as a control.

Figure 11 shows the results of 7.5% SDS-PAGE electrophoresis of the fractions purified in Example 6g. The meanings of the abbreviations used in Figure 11 are as follows:

M: Protein size standard (from top to bottom: 116kDa, 66kDa, 45kDa, 35kDa)

D: dialysate

N: unbound fraction

W: wash fraction

F1-F5 are eluted fractions containing plasminogen

Plg: plasminogen (American Diagnostica, Pfungstadt)

Example 6h: Evaluation of pH and substrate effects by fermentation of Pichia pastoris KM71/pAC37.1-3/1

Inoculate 2ml of Pichia pastoris KM71/pAC37.1-3/1 treated with glycerol at low temperature into 50ml of YEP-G medium (10 g/L yeast extract, 20 g/L casein peptone, 20 g/L Glycerol), the YEP-G medium was in a 1 liter wide neck shake flask without Schikanes. Cultivate for 9 hours at 30°C and 300 rpm. 5ml of this culture was then inoculated into 50ml MG medium [5 g/L yeast nitrogenous base (nitrgen base) without amino acids, 20 g/L glycerol, 2.5 mL/L biotin solution (0.2 g/L l)], the MG medium was in a 1 liter wide neck shake flask without Schikanes. The second preculture was cultured at 30° C. and 300 rpm for 16 hours. The main culture was fermented in a "stirrer-pro" (DASGIP, Jülich) multifermenter, a setup that allows simultaneous fermentation under 4 different conditions. Therefore, 15 ml of the second preculture was inoculated into 150 ml of BSM medium (see Example 6e). After a certain dose of substrate was added, the fermentation was started at pH = 6 and an effort was made to maintain this pH. The results of the fermentation carried out in parallel under different culture conditions are shown in Table 1:

Table 1: experiment pH substrate supply speed _OD600 Plasminogen concentration IIIIIIIV 6766 Methanol Methanol Methanol/Glycerin Methanol Portfolio Portfolio 1ml/hour Portfolio 187160270130 1.4 mg/L 6.1 mg/L 10.1 mg/L 3.4 mg/L

30 g/L of peptone was added to the medium of experiment IV. Before methanol begins to act, add glycerol-supply medium [500 g/L anhydrous glycerol, 10 mL/L trace element solution, 10 mL/L biotin solution (see Example 6e)] at 24 mL/hour Constant speed for 4 hours. The item combinations in Experiments I, II and IV were determined by the following formula as a dose function, f(x)=P1+(P2/1+exp(-P3(t-P4))))+(P5/(1 +exp(-P6(t-P7)))), where P1=0, P2=0.7, P3=0.2, P4=15, P5-P6=P7=0. As can be seen from Table 1, the highest concentration of plasminogen was obtained at neutral pH and mixed doses of methanol and glycerol.

Example 7a: Obtaining the lysine plasminogen gene from pAC37.1 and inserting it into pGAPZaA

150 ng of vector pGAPZαA (Invitrogen, Groningen, Netherlands) was digested with 10 U of restriction enzymes XhoI and NotI (both from Roche Diagnostics, Mannheim). At the same time, 300 ng of plasminogen expression vector pAC37.1 (see Example 6a) was digested with these two enzymes. The digested DNA fragments were separated by gel electrophoresis with 0.9% agarose. The 2715-base (bp) long plasminogen gene fragment obtained after pAC37.1 digestion and the 3073-base long fragment obtained after pGAPZαA digestion were purified with a QIAgen gel purification kit (Qiagen , Hilden). The two fragments were then mixed and ligated overnight at 4°C with 1 U of T4 DNA ligase.

Escherichia coli DH5α was transformed, and the purification and identification methods of the obtained plasmid were similar to those in Example 1b. However, ampicillin was used instead of melin to select transformants. The constructed plasmid was named pJW9.1 (Fig. 7).

Example 7b: Transformation of Pichia pastoris with plasmid pJW9.1

As in the method for transforming Pichia pastoris KM71H strain with pMHS476.1 in Example 1c, transform Pichia pastoris KM71H strain with plasmid pJW9.1, and linearize pJW9.1 with endonuclease BlnI before transformation change. The transformed cells were plated on YPDS-agar (EasySelect ^™ Pichia Expression Kit Instruction Manual) containing 100 μg/ml Meiyiling and cultured. The resulting clone was named Pichia pastoris KM71H/pJW9.1-a, where "a" still represents the serial numbering of the clone starting from 1.

Example 7c: Evaluation of pH value at different glycerol supply rates by fermentation of Pichia pastoris KM71H/pJW9.1-3

See Example 6i for the preculture and fermentation culture in "stirrer-pro", and see Table 2 for the results obtained.

Table 2: experiment pH substrate supply speed _OD600 Plasmin concentration IIIIIIIV 6.57.06.57.0 Glycerin Glycerin Glycerin Glycerin 1ml/hour 1ml/hour 0.5ml/hour 0.5ml/hour 22020314299 18.6 mg/L 22.2 mg/L 10.1 mg/L 3.8 mg/L

Again, the best results were obtained by fermentation at neutral pH, and the effect of substrate dosage (feed rate) on product formation is also clear from the table.

Example 7d: Fermentation of Pichia pastoris KM71H/pJW9.1-3

Pichia pastoris KM71H/pJW9.1-3 was inoculated into 50 ml of YEP-G medium (10 g/L yeast extract, 20 g/L casein, 20 g/L glycerol), the YEP -G medium was cultured in a 1-liter wide-necked flask without Schikanes at 30°C and 300 rpm for 9 hours. 10 ml of said culture was then inoculated into 40 ml of MG medium (5 g/l yeast nitrogenous base without amino acids, 20 g/l glycerol, 2.5 ml/l biotin solution (0.2 g/l), The MG medium was in a 1 liter wide-neck flask without Schikanes. The culture was grown at 30° C. and 300 rpm for 16 hours.

30 ml of the above-mentioned culture were inoculated into 3 liters of BSM-medium (see Example 6e) and then fermented in a 7.5 liter fermenter (type Labfors, Infors AG, CH). The fermentation temperature was 25°C, and the constant gas supply rate was 3.2 liters/minute. After 24 hours a glycerol solution [500 g/l glycerol, 10 ml/l trace element solution, 10 ml/l biotin solution (see Example 6e)] was added. During the fermentation, the rate of dosage was gradually increased from 10 ml/h to 45 ml/h. After 250 hours, the activity of 1375U/L plasminogen can be measured under the condition of streptokinase activation.

Example 8: Identification of plasminogen activators

The plasminogen-activating activity of 24 commercially available proteases was determined. The experimental reaction system is: 100 mM sodium phosphate buffer solution with pH=8.0, 0.36 mM CaCl ₂ , and 0.9% NaCl.

If the protease is provided as a powder, dissolve it in the buffer; if it is a solution, use it directly and dilute it with buffer as needed. 25 μl of protease solution was mixed with 25 μl of plasminogen solution (20 mg/ml) of the present invention and incubated at 37° C. for 10 minutes. The activity of plasmin was then determined by hydrolyzing the substrate N-tosyl-Gly-Pro-Lys-pNA. The specific method is: get 200 μl of substrate solution [9.5 mg of N-tosyl-Gly-Pro-Lys-pNA dissolved in 75 mg of glycine/10 ml and 2% Tween 20] into 850 μl of buffer, and then mix with Mix 50 μl of the previously incubated plasminogen-protease solution and continue to incubate at 37°C. The degree of increase in consumption can be determined by measuring the light absorption value at 405 nm. Consumption is due to proteases. A similar preincubated buffer and protease mixture was used instead of the preincubated plasmin and protease mixture at assay consumption.

Streptomyces griseus (S. griseus) protease, protease VIII, protease XXIII, protease XIX, protease XVIII, ficin, metalloendopeptidase, clostripain, Glu-C endoprotease, protease XIII, papain, chymotrypsin, Protease X, bromelain, vasodilation and protease A were purchased from Sigma, Deisenhofen; trypsin, papain, Asp-N endoprotease, neutral protease I, Lys-C endoprotease, thrombin and elastase were purchased from Roche, Mannheim; proteinase K from QIAGEN, Hilden. The concentration of each protease stock solution prepared is shown in Table 3. The dilution factor F indicates the factor by which the protease stock solution was diluted for the assay.

After activation, the following plasmin activities can be determined (1 U/mg = conversion of 1 μmol of N-tosyl-Gly-Pro-Lys-pNA per mg of protein per minute):

table 3: protease Activated plasmin activity Protein concentration (mg/ml) f Streptomyces griseus Protease Protease VIII Protease XXIII Protease XIX Protease XVIII Ficin Metalloendopeptidase Clostripain Glu-C Endoprotease Protease XIII Chymopapain Chymotrypsin Protease X Bromelain Protease A Trypsin Papain Asp-N Protease Neutral Protease ILys-C Endoprotease Thrombin Elastase Proteinase K 613.3U/mg9U/mg**0.7U/mg0.01U/mg8.9U/mg1.7U/mg0.6U/mg0.01U/mg*****0.02U/mg11kU/mg*4.3U/mg** 83.0U/mg0.63U/mg* 0.773.5817.82.781.790.810.010.250.810.432.020.142.010.810.560.363.400.640.0040.20.010.590.363.60 10001000500001001001111111111100000101115005100

*For the following enzymes, no plasminogen activity was detected: Protease XXIII, Protease XIX, Chymopapain, Lys-C Endoprotease, Chymotrypsin, Papain, Dispase I, Protease X, Bromelain, Shu Angiotin and proteinase K.

Embodiment 9: pharmaceutical preparation

The recombinant functional plasminogen used in the following examples is all obtained by the innovative method of the present invention. In this sense, "plasminogen" refers to recombinant micro-, small-, lysine- and glutamate plasminogens, while "plasmin" refers to proteolytically cleaved recombinant micro-, Plasmin derived from small-, lysine-, and glutamic acid plasminogen. Micro-, small-, lysine- and glutamate plasminogens can be activated with the same plasminogen activators, especially but not limited to the plasminogen activating proteases described above ; The ratio of activator and plasminogen (micro-, small-, lysine- and glutamate plasminogen) is about 1:1000.

Can activate plasminogen by proteolytic method, promptly these proteases mentioned in this patent application: as the urokinase of tissue type plasminogen activator, or proteases such as protease VIII, streptomyces griseus protease; Also can pass and Streptokinase or staphylokinase forms a complex, thereby activating plasminogen.

Example 9a: Pharmaceutical formulations

Hydrogels

Basic components of hydrogel (100g)

Plasminogen 100U

Plasminogen Activator 0.1U

Hydroxyethylcellulose 10000 3.5 grams

Optional components (sorbic acid/potassium sorbate 0.1-0.4%, PHB ester 0.1%)

Add purified water to 100.0g

Optionally, hypromellose or methyl cellulose can be used instead of hydroxyethyl cellulose, and the amount of hypromellose or methyl cellulose is 0.5-15.0 g.

gel

Plasminogen 1000U

Plasminogen Activator 1U

Glycerin (85%) 150.0 g

Hydroxyethyl Cellulose 10000 32.5g

Optional components (sorbic acid/potassium sorbate 0.1-0.4%, PHB ester 0.1%)

Lactate-free Ringer's solution up to 1000.0 g

or it could be:

100 g of gel contains:

Plasminogen 100U

Plasminogen Activator 0.1U

Hydroxyethylcellulose 30000 2.5 grams

Glycerin (85%) 10.0 g

Optional components (sorbic acid/potassium sorbate 0.1-0.2%, PHB ester 0.1%)

Add purified water to 100.0g

or it could be:

100 g of gel contains:

Plasminogen 100U

Plasminogen Activator 0.1U

polyacrylic acid 1 g

Propylene glycol 8 grams

Medium poly triglycerides 8 grams

Diethylamine (adjust pH) Appropriate amount

Optional components (sorbic acid/potassium sorbate 0.1-0.2%, PHB ester 0.1%)

2-propanol 0-1g

Add water to 100g

Hydrophilic ointment (polyethylene glycol ointment)

50g contains

Plasminogen 50U

Plasminogen Activator 0.05U

Macrogol 400 30.0 g

Macrogol 4000 10.0 g

Optional components (sorbic acid/potassium sorbate 0.1-0.2%, PHB ester 0.1%)

Add purified water to 50.0g

or it could be:

Anhydrous polyethylene glycol ointment

100 g contains

Plasminogen 100U

Plasminogen Activator 0.1U

Macrogol 300 50g

Macrogol 1500 Add to 100g

or it could be:

absorbent ointment

Plasminogen 100U

Plasminogen Activator 0.1U

Cetyl stearyl alcohol 29 g

Paraffin, viscous 34 g

Vaseline, white 100g

Hydrophobic ointment

Plasminogen 100U

Plasminogen Activator 0.1U

Vaseline 80.0g

Add dilute liquid paraffin to 100g

Hydrophobic ointment

Plasminogen 100U

Plasminogen Activator 0.1U

Hypromellose 400 20 grams

Petroleum jelly, white Add to 100g

or it could be:

Plasminogen 100U

Plasminogen Activator 0.1U

Carbomer (such as polyvinyl 974p) 15 grams

Paraffin, viscous 40 g

Petroleum jelly, white Add to 100g

Milk ester (creme)

Plasminogen 100U

Plasminogen Activator 0.1U

Medium poly triglycerides 20g

Emulsified Cetyl Stearyl Alcohol 10g

Lanolin 10 grams

Sorbitol 10g

Optional components (sorbic acid/potassium sorbate 0.1-0.2%, PHB ester 0.1%)

Add purified water to 100g

nonionic hydrophilic milk fat

Plasminogen 100U

Plasminogen Activator 0.1U

Palmitanol 20g

2-Ethyl myristate 10g

85% Glycerin 6 grams

Potassium sorbate 0.14 g

Citric acid 0.07 g

Add water to 100g

nonionic milk fat

Plasminogen 100U

Plasminogen Activator 0.1U

Polysorbate 60 5 g

Cetyl stearyl alcohol 10 g

85% Glycerin 10g

Vaseline, white 25 grams

Optional components (sorbic acid/potassium sorbate 0.1-0.2%, PHB ester 0.1%)

Add water to 100g

Liposome formulation

Plasminogen 100U

Plasminogen Activator 0.1U

Soy Lecithin, Chicken Lecithin 15g

Optional components (sorbic acid/potassium sorbate 0.1-0.2%, PHB ester 0.1% or diazobiphenylurea 1-2g)

Add water to 100.0g

capsule

One 0.25 g powder/granule capsule containing:

Plasminogen 5U

Plasminogen Activator 0.005U

Starch 0.1g

Silica 0.02g

Magnesium stearate 0.002 g

Polymethacrylate copolyester/polymethacrylate 0.015 g

Triethyl citrate 0.0005 g

Talc powder 0.001 g

microcrystalline cellulose up to 0.25 g

or it could be:

One 0.25 g powder/granule capsule containing:

Plasminogen 5U

Plasminogen Activator 0.005U

Silica 0.01g

Magnesium stearate 0.002 g

Polymethacrylate copolyester/polymethacrylate 0.015 g

Triethyl citrate 0.0001 g

Talc powder 0.001 mg

Mannitol up to 0.25 g

pill

100 mg granulated pills contain:

Plasminogen 5U

Plasminogen Activator 0.005U

Starch 30 mg

Silica 2 mg

Magnesium stearate 4 mg

Polymethacrylate copolyester/polymethacrylate 5 mg

Triethyl citrate 0-1 mg

Talc powder 0.00001 mg

Microcrystalline Cellulose up to 100mg

small pill

100 g small pills contain

Plasminogen 2000U

Plasminogen Activator 2U

Starch 20 grams

Sucrose stearate 20g

Silica 2 grams

Magnesium stearate 3 grams

Polyvinylpyrrolidone 0-1g

Polymethacrylate copolyester/polymethacrylate 5 g

Talc powder 0.2 g

Triethyl citrate 0.1g

microcrystalline cellulose up to 100 g

injection

Plasminogen 500U

Plasminogen Activator 0.5U

Ethanol 0-1g

Propylene glycol 10 grams

Polyethylene glycol 0-1g

Sodium Chloride Appropriate amount

Optional buffer (disodium hydrogen phosphate/sodium dihydrogen phosphate buffer)

Add purified water to 100ml

In such formulations, instead of micro-, mini-, lysine- or glutamate-plasminogen, plasminogen can be used in an amount producing the same activity. If plasmin is used directly, then the drug formulation does not need to contain a plasminogen activator.

Example 9b: Pharmaceutical formulations

a) Hydrogel

Basic components of hydrogel (100g)

Plasmin 100U

Hydroxyethylcellulose 400 2.5-5 grams

Add purified water to 100.0g

The swelling time takes 1 to 3 hours.

Each gram of hydrogel can contain 1-1000 U of plasmin.

b) Hydrophilic ointment

Basic composition of hydrophilic ointment (1000g)

Plasmin 1000U

Anhydrous glycerin 85.0g

Hydroxyethyl Cellulose 10000 32.5g

Optionally add inositol polynicotinate (polyhexanide) 2% by weight

Lactate-free Ringer's solution up to 1000.0 g

Inositol polynicotinate can be optionally added as an active substance to inhibit the growth of microorganisms, but not more than 0.2% of the total weight. Hydroxyethylcellulose 400 (such as: TyloseH 300 or Natrosol 250HX PHARM) can be added to replace hydroxyethylcellulose 10000 (Natrosol 250HXPHARM).

Each gram of ointment can contain 1-10000U of plasmin.

c) ointment

Basic composition of ointment (50g)

Plasmin 50U

Macrogol 400 30.0-32.5 grams

Macrogol 400 012.5-7.5 grams

Add purified water to 50.0g

preparation:

Mix 12.5 grams of polyethylene glycol 4000 and 30.0 grams of polyethylene glycol 400 (7.5 grams of polyethylene glycol 4000 and 32.5 grams of polyethylene glycol 400 can also be used) into the ointment tray, add hot water to the bath until the polyethylene glycol Alcohol melts. After cooling, an appropriate amount of plasminogen produced according to the method of the present invention can be added, which has been dissolved in 7.5 g of purified water, and then homogenized.

d) Capsules

Basic composition of 0.5 g capsule

Plasminogen 5U

Lactose 0.42 grams

Starch 0.06 g

Magnesium stearate 0.02 g

Each capsule can contain 0.1-100U of plasmin.

e) injection/infusion

Basic composition of 100ml

Plasmin 500U

Ethanol 0.01g

Propylene Glycol 30ml

Add purified water to 100ml

The solution may contain 1-500 U of plasmin per ml.

Micro-, small-, lysine-, glutamic acid-plasminogen can be used to replace plasminogen in the above-mentioned active unit amount, if at least one plasminogen activator is added at the same time, the added fibrinolytic The ratio of zymogen activator to plasminogen should be between 1:10000 and 1:100, preferably 1:1000, and the amount is the active amount of plasminogen.

Example 10a: Different forms of small plasminogen and microplasminogen genes were amplified and cloned into the vector pPICZaA; Pichia pastoris was transformed.

Small plasminogen, microplasminogen is a shorter derivative than full-length plasminogen, which lacks the N-terminal domain, but still has the activity of being activated into plasmin. In order to amplify and clone the small plasminogen and microplasminogen genes into the vector pPICZαA, the 3' end primer NO34 and different 5' end primers NO36a-j (sequence numbers No.19 to 28) were designed, Amplification conditions were the same as those described in Example 1a. In addition to the complementary bases with the plasminogen gene, the oligonucleotide primers NO36a, c, e, j, i also have the codons of the Kex2 restriction site; the primers NO36b, d, f, h, j, then Two codons for the restriction site for Ste13 were added downstream of the codon for the restriction site for Kex2. Primer NO34 also has a KspI restriction site, while primers NO36 a-j contain an XhoI restriction site.

The method for cloning the small plasminogen and microplasminogen genes into the vector pPICZαA is similar to the method in Example 1b, except that both the vector and the specific PCR product are digested with restriction endonucleases XhoI and KspI. The primers used, the names of the obtained plasminogen derivatives, the encoded protease cleavage sites, the names of the obtained plasmids and the names of the N-terminal amino acids of the secreted plasminogen derivatives are summarized in the table below. 5'-primer 3'-primer name protease-cleavage site Plasmid name N-terminal amino acid* NO36aNO36bNO36cNO36dNO36eNO36fNO36gNO36hNO36iNO36j NO34NO34NO34NO34NO34NO34NO34NO34NO34NO34 microplasminogen microplasminogen microplasminogen microplasminogen microplasminogen microplasminogen microplasminogen microplasminogen microplasminogen microplasminogen microplasminogen microplasminogen Kex2Kex2, 2 Ste13Kex2Kex2, 2 Ste13Kex2Kex2, 2 Ste13Kex2Kex2, 2 Ste13Kex2Kex2, 2 Ste13 pPLG1.1pPLG2.1pPLG3.2pPLG4.2pPLG5.3pPLG6.1pPLG7.1pPLG8.3pPLG9.1pPLG10.1 A463A463K550K550L551L551A562A562S564S564

* refers to preplasminogen (Seq. ID No.12) composed of 810 amino acids.

Figure 8 shows an example: plasmid pPLG1.1.

As in the method for transforming Pichia pastoris KM71H strain with pMHS476.1 in Example 1c, transform Pichia pastoris KM71H strain with pPLG1.1, and the clone obtained is named Pichia pastoris KM71H/pPLG1 .1-1/a, where "a" still represents the sequential numbering of the clones starting from 1.

According to the method for generating the KM71H/pPLG1.1-1/a strain, different strains were generated using the following plasmids: pPLG2.1, pPLG3.2, pPLG4.2, pPLG5.3, pPLG6.1, pPLG7.1, pPLG8.3 , pPLG9.1 or pPLG10.1.

Oligonucleotide primers NO36a-j:

NO36a AAAAACTCGAGAAAAGAGCACCCTCCGCCTGTTG

NO36b AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCACCTCCGCCTGTTG

NO36c AAAAACTCGAGAAAAGAAAACTTTACGACTACTG

NO36d AAAAACTCGAGAAAAGAGAGGCTGAAGCTAAACTTTACGACTACTG

NO36e AAAAACTCGAGAAAAGACTTTACGACTACTGTG

NO36f AAAAACTCGAGAAAAGAGAGGCTGAAGCTCTTTACGACTACTGTG

NO36g AAAAACTCGAGAAAAGAGCCCCTTCATTTGATTGTG

NO36h AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCCCCTTCATTTGATTGTG

NO36i AAAAACTCGAGAAAAGATCATTTGATTGTGGGAAGCC

NO36j AAAAACTCGAGAAAAGAGAGGCTGAAGCTTCATTTGATTGTGGGAAGCC

Example 10b: Different forms of the small-, micro-plasminogen gene were amplified and cloned into the vector pGAPZaA; it was transformed into Pichia pastoris.

In order to amplify and clone the small- and micro-plasminogen gene into the vector pGAPZαA, the 3' end primer NO34 and different 5' end primers NO36a-j (Seq.ID No.19 to 28) were designed. The increased conditions were identical to those described in Example 1a. Oligonucleotide primers NO36a, c, e, j, i have codons for Kex2 restriction sites next to the complementary bases of the plasminogen gene; primers NO36b, d, f, h, j have Kex2 restriction sites Downstream of the site codon, two codons for the restriction site of Ste13 were added. Primer NO34 also has a KspI restriction site, while primers NO36a-j contain XhoI restriction sites.

The method for cloning the small- and micro-plasminogen gene into the vector pGAPZαA is similar to the method in Example 1b, except that both the vector and the PCR product are digested with restriction endonucleases XhoI and KspI. The primers used, the names of the plasminogen derivatives, the encoded protease cleavage sites, the names of the resulting plasmids and the names of the N-terminal amino acids of the secreted plasminogen derivatives are summarized in the table below. 5'-primer 3'-primer name protease-cleavage site Plasmid name N-terminal amino acid* NO36aNO36bNO36cNO36dNO36eNO36fNO36gNO36hNO36iNO36j NO34NO34NO34NO34NO34NO34NO34NO34NO34NO34 microplasminogen microplasminogen microplasminogen microplasminogen microplasminogen microplasminogen microplasminogen microplasminogen microplasminogen microplasminogen microplasminogen microplasminogen Kex2Kex2, 2 Ste13Kex2Kex2, 2 Ste13Kex2Kex2, 2 Ste13Kex2Kex2, 2 Ste13Kex2Kex2, 2 Ste13 pPLG11.2pPLG11.2pPLG13.1pPLG14.2pPLG15.1pPLG16.3pPLG17.2pPLG18.1pPLG19.2pPLG20.1 A463A463K550K550L551L551A562A562S564S564

* refers to preplasminogen (Seq. ID No.12) composed of 810 amino acids.

Figure 9 shows an example: plasmid pPLG11.2.

As in the method for transforming Pichia pastoris KM71H strain with pJW9.1 in Example 7a, transform Pichia pastoris KM71H strain with pPLG11.2, and cut the plasmid with endonuclease BlnI before transformation to make it linear . The resulting clone was named Pichia pastoris KM71H/pPLG11.2-1/a, where "a" still represents the sequential numbering of the clone starting from 1.

According to the method for generating the KM71H/pPLG1.1-1/a strain, different strains were generated using the following plasmids: pPLG12.1, pPLG13.1, pPLG14.2, pPLG15.1, pPLG16.3, pPLG17.2, pPLG18.1 , pPLG19.2 and pPLG20.1.

sequence record

Sequence 1: oligonucleotide primer NO34

AAAAACCGCGGTCAATTATTTTCTCATCACTCCC

Sequence 2: oligonucleotide primer NO36

AAAAACTCGAGAAAAGAAAAGTGTATCTCTCAGAGTG

Sequence 3: oligonucleotide primer NO57

AAAAACTCGAGAAAAGAGAGGCTGAAGCTAAAAGTGTATCTCTCAGAGTG

Sequence 4: oligonucleotide primer NO37

AAAAATTCGAAAAATGGAACATAAGGAAGTGG

Sequence 5: oligonucleotide primer NO35

AAAAACTCGAGAAAAAGAGAGCCTCTGGATGACTAT

Sequence 6: oligonucleotide primer NO56

AAAAACTCGAGAAAAGAGAGGCTGAAGCTGAGCCTCTGGATGACTAT

Sequence 7: human lysine plasminogen fusion gene, which contains the code for Kex2 protease cleavage site

sub, and the gene for the signal sequence of Saccharomyces cerevisiae alpha factor

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT

CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT

TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT

AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA

TCTCTCGAGAAAAAAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTAC

AGAGGGACGATGTCCAAAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTTCT

CCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTAC

TGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAG

AGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAA

AACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCT

CAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAG

AATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCCAAC

AAGCGCTGGGAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTCCC

ACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTT

TCCGGGCACACCTGTCAGCACTGGAGTGCACAGACCCTCACACACATAACAGGACACCA

GAAAACTTCCCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAAAGG

GCCCCATGGTGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCC

TGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAACC

CCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCCACC

ACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAGAAG

ACCCCAGAAAACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGATGCC

GATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTG

AAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCA

GATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGC

AAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCAT

AGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAAATTACTGC

CGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTT

TACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAA

GTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCC

TGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTG

ATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCA

TCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAA

ATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTA

AGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTAT

GTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTT

GGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGC

TATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGA

GGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAA

TACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTz

GTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 8: human lysine plasminogen fusion protein, which contains Kex2 protease cleavage site and signal peptide of Saccharomyces cerevisiae alpha factor

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV

AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKRKVYLSECKTGNGKNY

RGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQG

PWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDS

QSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPR

CTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTP

ENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQ

LAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQK

TPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASV

VAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPH

RHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCA

APSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTL

ISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEEVSRLFLEP

TRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTF

GAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSG

GPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

Sequence 9: human lysine plasminogen fusion gene, which contains the codons of Kex2 protease cleavage site, two Ste3 protease cleavage sites, and the signal peptide sequence of Saccharomyces cerevisiae alpha factor

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT

CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT

TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT

AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA

TCTCTCGAGAAAAGAGAGGCTGAAGCTAAAAGTGTATCTCTCAGAGTGCAAGACTGGGAAT

GGAAAGAACTACAGAGGGACGATGTCCAAAAACAAAAAATGGCATCACCTGTCAAAAATGG

AGTTCCACTTCTCCCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTG

GAGGAGAACTACTGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACT

GATCCAGAAAAGAGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCAT

TGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAG

GCCTGGGACTCTCAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAG

AACCTGAAGAAGAATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACC

ACCGACCCCAAACAAGCGCTGGGAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCA

TCTTCTGGTCCCACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTG

GCTGTTACCGTTTCCGGGCACACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACAT

AACAGGACACCAAAAACTTCCCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCT

GACGGAAAAAGGGCCCCATGGTGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGT

AAGATACCGTCCTGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCCACAGCACCA

CCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGC

ACATCCTCCACCACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACAC

CGGCACCAGAAGACCCCAGAAAACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGG

AATCCAGATGCCGATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAG

TACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTT

GTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAA

GGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCC

CAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAA

AAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAAT

CCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGT

GGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCC

CACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGT

GGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCC

CCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCG

CATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCC

TTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCA

TCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACC

CAAGGTACTTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAA

GTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGG

CATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTC

GAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCC

AATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATG

AGAAATAATTGA

SEQUENCE 10: Human lysine plasminogen fusion protein, which contains Ste3, Kex2 protease cleavage sites, and signal peptide of Saccharomyces cerevisiae α factor.

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV

AVLPFSNSTNGLLFINTTIASIAAKEEGVSLEKREAEAKVYLSECKTGN

GKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDN

DPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQ

AWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELC

DIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTH

NRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPV

STEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPH

RHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGT

EASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAA

QEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDV

PQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFC

GGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEEVSRL

FLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGET

QGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQ

GDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVM

RNN*

SEQ ID NO: 11: human preplasminogen gene

ATGGAACATAAGGAAGTGGTTCTTCTACTTCTTTTATTTCTGAAATCAGGTCAAGGAGAG

CCTCTGGATGACTATGTGAATACCCAGGGGGCTTCACTGTTCAGTGTCACTAAGAAGCAG

CTGGGAGCAGGAAGTATAGAAGAATGTGCAGCAAAATGTGAGGAGGACGAAGAATTCACC

TGCAGGGCATTCCAATATCACAGTAAAGAGCAACAATGTGTGATAATGGCTGAAAACAGG

AAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTTGAAAAGAAAGTGTATCTC

TCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCCAAAAACAAAAAAT

GGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGACCTAGATTCTCACCTGCT

ACACACCCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGACAACGATCCGCAG

GGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGCGACATTCTTGAG

TGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAGACC

ATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCACACGCTCATGGATACATT

CCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGTCGTAACCCCGATAGGGAG

CTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGGGAACTTTGCGACATCCCC

CGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAGTGTCTGAAGGGAACAGGT

GAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCACACCTGTCAGCACTGGAGT

GCACACCACCCCTCACACACATAACAGGACACCAAAACTTCCCCTGCAAAAATTTGGAT

GAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGGTGCCATACAACCAACAGC

CAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGTGACTCCTCCCCAGTATCCACGGAA

CAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCATGGT

GATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAGAAGTGTCAGTCT

TGGTCATCTATGACACCCACACCGGCACCAGAAGACCCCAGAAAACTACCCAAATGCTGGC

CTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGGTGTTTTACCACA

GACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCGAGT

GTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGAC

TGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACG

CCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACA

AATCCACGGGCGGGTCTGGAAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGT

CCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGT

GCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGG

GTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACA

AGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCT

GCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACAC

CAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAG

CCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAA

GTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTC

ATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAG

CTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAA

TCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGT

GGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGG

GGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTT

ACTTGGATTGAGGGAGTGATGAGAAATAATTGA

SEQUENCE 12: Human preplasminogen

MEHKEVVLLLLLFLKSGQGEPLDDYVNTQGASLFSVTKKQLGAGSIEECA

AKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYL

SECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEEN

YCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKT

MSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDP

NKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWS

AQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIP

SCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQS

WSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCN

LKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGT

PCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRK

LYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRT

RFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQ

EIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECF

ITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLA

GGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFV

TWIEGVMRNN*

SEQ ID NO: 13: human glutamic acid plasminogen fusion gene, which contains the codon of Kex2 protease cleavage site, and the signal sequence of Saccharomyces cerevisiae alpha factor

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT

CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT

TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT

AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA

TCTCTCGAGAAAAGAGAGCCTCTGGATGACTATGTGAATACCCAGGGGGCTTCACTGTTC

AGTGTCACTAAGAAGCAGCTGGGAGCAGGAAGTATAGAAGAATGTGCAGCAAAATGTGAG

GAGGACGAAGAATTCACCTGCAGGGCATTCCAATATCACAGTAAAGAGCAACAATGTGTG

ATAATGGCTGAAAACAGGAAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTT

GAAAAGAAAGTGTATCTCTCGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACG

ATGTCCAAAAAAAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGA

CCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAAT

CCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGAC

TACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGAC

GGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCA

CACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGT

CGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCCAACAAGCGCTGG

GAACTTTGCGACATCCCCCGCTGCACAAACACCTCCACCATCTTCTGGTCCCACCTACCAG

TGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCAC

ACCTGTCAGCACTGGAGTGCACAGACCCTCACACACATAACAGGACACCAGAAAACTTC

CCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGG

TGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCC

TCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTC

CAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCCCACCACCACCACA

GGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCCACACCGGCACCAGAAGACCCCAGAA

AACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGC

CCCTGGTGTTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGC

TCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAG

ACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCG

ACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGC

ATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAAATTACTGCCGTAACCCT

GATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTAC

TGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCG

AAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGG

CAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCA

GAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAG

GTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTG

TCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCT

GCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCT

GACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGC

CTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTT

CTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGAC

AGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTA

CAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTT

CGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

SEQUENCE 14: Human glutamic acid plasminogen fusion protein, which contains Kex2 protease cleavage site, and signal peptide of Saccharomyces cerevisiae alpha factor

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV

AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREPLDDYVNTQGASLF

SVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSI

IIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHR

PRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEE

CMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYC

RNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYR

GNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPW

CHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQS

YRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKG

PWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFG

NGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNP

DGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGG

CVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYK

VILGAHQEVNLEPHVQEIEEVSRLFLEPTRKDIALLKLSSPAVITDKVIPA

CLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEF

LNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGC

ARPNKPGVYVRVSRFVTWIEGVMRNN*

SEQ ID NO: 15: human glutamic acid plasminogen fusion gene, which contains the codons of Kex2, two Ste13 protease cleavage sites, and the signal peptide gene of Saccharomyces cerevisiae alpha factor

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT

CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT

TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT

AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA

TCTCTCGAGAAAAGAGAGGCTGAAGCTGAGCCTCTGGATGACTATGTGAATACCCAGGGG

GCTTCACTGTTCAGTGTCACTAAGAAGCAGCTGGGAGCAGGAAGTATAGAAGAATGTGCA

GCAAAATGTGAGGAGGACGAAGAATTCACCTGCAGGGCATTCCAATATCACAGTAAAGAG

CAACAATGTGTGATAATGGCTGAAAACAGGAAGTCCTCCATAATCATTAGGATGAGAGAT

GTAGTTTTATTTGAAAAGAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAAC

TACAGAGGGACGATGTCCAAAAAAAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACT

TCTCCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAAC

TACTGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAA

AAGAGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGA

GAAAACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGAC

TCTCAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAG

AAGAATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCC

AACAAGCGCTGGGAACTTTGCGACATCCCCCGCTGCACAAACACCTCCACCATCTTCTGGT

CCCACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACC

GTTTCCGGGCACACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACA

CCAGAAAACTTCCCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAA

AGGGCCCCATGGTGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCG

TCCTGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTA

ACCCCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCC

ACCACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCCACACCGGCACCAG

AAGACCCCAGAAAACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGAT

GCCGATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAAC

CTGAAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTT

CCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGA

GGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCC

CATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTAC

TGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAA

CTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCT

CAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACAT

TCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACC

TTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCT

TCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAG

GAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAG

CTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAAT

TATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACT

TTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAAT

CGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCC

GGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGAC

AAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCT

GGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAAT

TGA

Sequence 16: human glutamic acid plasminogen fusion protein, which contains Kex2, Ste13 restriction sites, and the signal peptide of Saccharomyces cerevisiae alpha factor

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV

AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEAEPLDDYVNTQG

ASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENR

KSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSST

SPHRRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILE

CEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLK

KNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTG

ENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGK

RAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHG

DGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPD

ADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEED

CMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNY

CRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGR

VVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRP

SSYKVILGAHQEVNLEPHVQEIEEVSRLFLEPTRKDIALLKLSSPAVITDK

VIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCN

RYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSW

GLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

SEQ ID NO: 17: Sequence of plasminogen glutamate secreted into culture medium (pSM49.8, pSM58.1 and pSM82.1)

EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSK

EQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTK

NGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDP

EKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGY

IPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTTPPPSS

GPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNL

DENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPE

LTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNA

GLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVL

LPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFPE

TNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGK

PQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLT

AAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEEVSRLFLEPTRKDIALL

KLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEA

QLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEK

DKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

SEQ ID NO: 18: Protein sequence of lysine plasminogen secreted into the medium (pMHS476.1, pSM54.2, pAC37.1 and pJW9.1).

KVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEG

LEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGK

ISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCF

TTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTC

QHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEY

CKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGK

KCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRW

EYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATT

VTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTT

NPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQV

SLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLE

PHVQEIEEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADR

TECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCA

GHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRV

SRFVTWIEGVMRNN*

SEQ ID NO: 19: oligonucleotide primer NO36a

AAAAACTCGAGAAAAGAGCACCTCCCGCCTGTTG

SEQ ID NO: 20: oligonucleotide primer NO36b

AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCACCTCCGCCTGTTG

SEQ ID NO: 21: oligonucleotide primer NO36c

AAAAACTCGAGAAAAGAAAACTTTACGACTACTG

SEQ ID NO: 22: oligonucleotide primer NO36d

AAAAACTCGAGAAAAGAGAGGCTGAAGCTAAACTTTACGACTACTG

SEQ ID NO: 23: oligonucleotide primer NO36e

AAAAACTCGAGAAAAGACTTTACGACTACTGTG

SEQ ID NO: 24: oligonucleotide primer NO36f

AAAAACTCGAGAAAAGAGAGGCTGAAGCTCTTTACGACTACTGTG

SEQ ID NO: 25: oligonucleotide primer NO36g

AAAAACTCGAGAAAAGAGCCCCTTCATTTGATTGTG

SEQ ID NO: 26: oligonucleotide primer NO36h

AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCCCCTTCATTTGATTGTG

SEQ ID NO: 27: oligonucleotide primer NO36i

AAAAACTCGAGAAAAGATTCATTTGATTGTGGGAAGCC

SEQ ID NO: 28: oligonucleotide primer NO36j

AAAAACTCGAGAAAAGAGAGGCTGAAGCTTCATTTGATTGTGGGAAGCC

SEQ ID NO: 29: Small plasminogen (pPLG1.1 and pPLG2.1)

APPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHR

HSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAA

PSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLI

SPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEEVSRLFLEPT

RKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFG

AGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGG

PLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

SEQ ID NO: 30: Microplasminogen (pPLG3.2 and pPLG4.2)

KLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLR

TRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHV

QEIEEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTEC

FITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHL

AGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRF

VTWIEGVMRNN*

SEQ ID NO: 31: Microplasminogen (pPLG5.3 and pPLG6.1).

LYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRT

RFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQ

EIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECF

ITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLA

GGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFV

TWIEGVMRNN*

SEQ ID NO: 32: Microplasminogen (pPLG7.1 and pPLG8.3).

APSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTL

ISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEEVSRLFLEP

TRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTF

GAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSG

GPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

SEQ ID NO: 33: Microplasminogen (pPLG9.1 and pPLG10.1).

SFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLIS

PEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEEVSRLFLEPTR

KDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGA

GLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGP

LVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

SEQ ID NO: 34: DNA sequence of Saccharomyces cerevisiae α factor in pPICZαA, which is located upstream of the Kex2 restriction site

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAG

SEQ ID NO: 35: Amino acid sequence of Saccharomyces cerevisiae alpha factor in pPICZαA located upstream of the Kex2 restriction site

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLE

SEQ ID NO: 36: DNA sequence of Kex2 restriction site

AAAAGA

SEQ ID NO: 37: DNA sequence of Ste13 restriction site

GAGGCTGAAGCT

SEQ ID NO: 38: Amino acid sequence of Kex2 restriction site

KR

SEQ ID NO: 39: Amino acid sequence of Ste13 restriction site

EAEA

SEQ ID NO: 40: Amino acid sequence consisting of human small plasminogen in plasmid pPLG1.1, Kex2 restriction site and propeptide of Saccharomyces cerevisiae alpha factor

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV

AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKRAPPVVLLPDVETPS

EEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLE

KNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKC

PGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKS

PRPSSYKVILGAHQEVNLEPHVQEIEEVSRLFLEPTRKDIALLKLSSPAVI

TDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENK

VCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGV

TSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

SEQ ID NO: 41: Amino acid sequence consisting of human small plasminogen in plasmid pPLG2.1, Kex2 restriction site, two Ste1 restriction sites and propeptide of Saccharomyces cerevisiae alpha factor

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV

AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEAAPPPVVLLPDV

ETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPR

AGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVE

PKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHC

LEKSPRPSSYKVILGAHQEVNLEPHVQEIEEVSRLFLEPTRKDIALLKLSS

PAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPV

IENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYI

LQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

SEQ ID NO: 42: Amino acid sequence consisting of: human microplasminogen in plasmid pPLG3.2, Kex2 restriction site and propeptide of Saccharomyces cerevisiae alpha factor

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLEKRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPH

SWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIE

VSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLL

KEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTS

WGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

SEQ ID NO: 43: Amino acid sequence consisting of human microplasminogen in plasmid pPLG4.2, Kex2 restriction site, two Ste13 restriction sites and prepro-peptide of Saccharomyces cerevisiae alpha factor

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLEKREAEAKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCV

AHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHV

QEIEEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFG

AGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQ

GVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

SEQ ID NO: 44: Amino acid sequence consisting of: human microplasminogen in plasmid pPLG5.3, Kex2 restriction site and propeptide of Saccharomyces cerevisiae alpha factor

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLEKRLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHS

WPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEV

SRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLK

EAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSW

GLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

SEQ ID NO: 45: Amino acid sequence consisting of: human microplasminogen in plasmid pPLG6.1, Kex2 restriction site, two Ste13 restriction sites and propeptide propeptide of Saccharomyces cerevisiae alpha factor

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLEKREAEALYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVA

HPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQ

EIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGA

GLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQG

VTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

SEQ ID NO: 46: Amino acid sequence consisting of: human microplasminogen in plasmid pPLG7.1, Kex2 restriction site and propeptide of Saccharomyces cerevisiae alpha factor

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLEKRAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRF

GMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEEVSRLFLEPTRKD

IALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKV

CNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPG

VYVRVSRFVTWIEGVMRNN*

SEQ ID NO: 47: Amino acid sequence consisting of human microplasminogen in plasmid pPLG8.3, Kex2 restriction site, two Ste13 restriction sites and propeptide propeptide of Saccharomyces cerevisiae alpha factor

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLEKREAEAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSL

RTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEP

TRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVI

ENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARP

NKPGVYVRVSRFVTWIEGVMRNN*

SEQ ID NO: 48: Amino acid sequence consisting of: human microplasminogen in plasmid pPLG9.1, Kex2 restriction site and propeptide of Saccharomyces cerevisiae alpha factor

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLEKRSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGM

HFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEEVSRLFLEPTRKDIA

LLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCN

RYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVY

VRVSRFVTWIEGVMRNN*

SEQ ID NO: 49: Amino acid sequence consisting of human microplasminogen in plasmid pPLG10.1, Kex2 restriction site, two Ste13 restriction sites and propeptide propeptide of Saccharomyces cerevisiae alpha factor

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLEKREAEASFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRT

RFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTR

KDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIEN

KVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNK

PGVYVRVSRFVTWIEGVMRNN*

SEQ ID NO: 50: Nucleic acid sequence consisting of the following sequences: the human small plasminogen gene in the plasmid pPLG1.1, the codon of the Kex2 restriction site and the gene of Saccharomyces cerevisiae α factor prepro-peptide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATG

TTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAG

GACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCG

GGTCTGGAAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACG

ACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGAT

TGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCC

CACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGA

GGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGG

CCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAG

GAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTA

AGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTG

GTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCT

GGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTT

CTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGT

TGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGA

GTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCA

AGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

SEQ ID NO: 51: a nucleic acid sequence consisting of the following sequences: the human small plasminogen gene in the plasmid pPLG2.1, the codons of the Kex2 restriction site and the Ste13 restriction site, and the gene of Saccharomyces cerevisiae α factor prepro-peptide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGAGAGGCTGAAGCTGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAA

GAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGG

ACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACA

AATCCACGGGCGGGTCTGGAAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCCC

TGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCC

CCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGG

GGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATG

CACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAG

AAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAA

CCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCC

TTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCC

CCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGT

ACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAAT

CGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGA

GGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATAC

ATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTAT

GTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

SEQ ID NO: 52: Nucleic acid sequence consisting of the human microplasminogen gene in plasmid pPLG3.2, the codon of Kex2 restriction site and the gene of Saccharomyces cerevisiae alpha factor prepro-pro-peptide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAG

CCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACAT

TCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTG

ATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCC

TACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAA

GTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCT

GCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGAC

CGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTC

AAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGA

AGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGT

GACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCT

TGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTT

ACTTGGATTGAGGGAGTGATGAGAAATAATTGA

SEQ ID NO: 53: Nucleic acid sequence consisting of the human microplasminogen gene in plasmid pPLG4.2, the codons of Kex2 restriction site and Ste13 restriction site, and the gene of Saccharomyces cerevisiae alpha factor prepro-pro-peptide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGAGAGGCTGAAGCTAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTT

GATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTG

GCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGT

GGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCA

AGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTT

CAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAG

CTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTAT

GTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGA

GCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAG

TTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGAC

AGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAA

GGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTT

TCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

SEQ ID NO: 54: Nucleic acid sequence consisting of the human microplasminogen gene in the plasmid pPLG5.3, the codon of the Kex2 restriction site and the gene of Saccharomyces cerevisiae alpha factor prepro-pro-peptide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCT

CAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCC

TGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATA

TCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTAC

AAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTG

TCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCC

GTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGG

ACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTTGGAGCTGGCCTTCTCAAG

GAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGA

GTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGAC

AGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGG

GGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACT

TGGATTGAGGGAGTGATGAGAAATAATTGA

SEQ ID NO: 55: a nucleic acid sequence consisting of the human microplasminogen gene in the plasmid pPLG6.1, the codons of the Kex2 restriction site and the Ste13 restriction site, and the gene of Saccharomyces cerevisiae alpha factor prepro-peptide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGAGAGGCTGAAGCTCTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGAT

TGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCC

CACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGA

GGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGG

CCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAG

GAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTA

AGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTG

GTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCT

GGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTT

CTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGT

TGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGA

GTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCA

AGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

SEQ ID NO: 56: Nucleic acid sequence consisting of the human microplasminogen gene in plasmid pPLG7.1, the codon of Kex2 restriction site and the gene of Saccharomyces cerevisiae alpha factor prepro-pro-peptide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGAGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTT

GTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTT

GGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGC

TTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAAT

CTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGAT

ATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTG

CCATCCCCAAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACC

CAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTG

TGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTG

GCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGAC

AAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGT

GTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

SEQ ID NO: 57: Nucleic acid sequence consisting of the human microplasminogen gene in plasmid pPLG8.3, the codons of Kex2 restriction site and Ste13 restriction site, and the gene of Saccharomyces cerevisiae α factor prepro-pro-peptide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGAGAGGCTGAAGCTGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGT

CCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTT

AGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACT

GCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACAC

CAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCC

ACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATC

CCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGC

TGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATT

GAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGT

GCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGC

TTCGAGAAGGACAAATACATTTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCC

AATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGA

AATAATTGA

SEQ ID NO: 58: Nucleic acid sequence consisting of the human microplasminogen gene in plasmid pPLG9.1, the codon of Kex2 restriction site and the gene of Saccharomyces cerevisiae alpha factor prepro-pro-peptide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGATCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGG

GGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATG

CACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAG

AAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAA

CCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCC

TTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCC

CCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGT

ACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAAT

CGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGA

GGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATAC

ATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTAT

GTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

SEQ ID NO: 59: Nucleic acid sequence consisting of the human microplasminogen gene in plasmid pPLG10.1, the codons of the Kex2 restriction site and the Ste13 restriction site, and the gene of Saccharomyces cerevisiae alpha factor

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGAGAGGCTGAAGCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGA

AGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACA

AGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCC

CACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAA

GTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGA

AAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCT

TGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGA

GAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAAT

AAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGG

CATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAG

AAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAG

CCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAAT

TGA

SEQ ID NO: 60: Nucleic acid sequence of human small plasminogen gene in plasmids pPLG1.1 and pPLG2.1.

GCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTT

GGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGAC

TGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGT

CTGGAAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACA

AATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGT

GGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCAC

CCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGC

ACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCT

TCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAA

ATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGC

AGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTC

GCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGC

CTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTG

AATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGC

CAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTC

ACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGG

TTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

SEQ ID NO: 61: Nucleic acid sequence of human microplasminogen gene in plasmids pPLG3.2 and pPLG4.2

AAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCT

CAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCC

TGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATA

TCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTAC

AAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTG

TCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCC

GTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGG

ACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTTGGAGCTGGCCTTCTCAAG

GAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGA

GTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGAC

AGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGG

GGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACT

TGGATTGAGGGAGTGATGAGAAATAATTGA

SEQ ID NO: 62: Nucleic acid sequence of human microplasminogen gene in plasmids pPLG5.3 and pPLG6.1

CTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAA

GTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGG

CCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCC

CCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAG

GTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCT

AGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTC

ATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACC

GAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAA

GCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTC

CAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGT

GGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGT

CTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGG

ATTGAGGGAGTGATGAGAAATAATTGA

SEQ ID NO: 63: Nucleic acid sequence of human microplasminogen gene in plasmids pPLG7.1 and pPLG8.3

GCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTG

GGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGA

ATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTG

GAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTC

GAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATT

GCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCA

TCCCCAAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAA

GGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGC

AATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCC

GGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAA

TACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTC

TATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

SEQ ID NO: 64: Nucleic acid sequence of human microplasminogen gene in plasmids pPLG9.1 and pPLG10.1

TCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGG

TGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCAC

TTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAG

TCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCG

CATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTG

CTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCA

AATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACT

TTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGC

TATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGC

ACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATT

TTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTT

CGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

SEQ ID NO: 65: Nucleic acid sequence of human glutamate plasminogen gene

GAGCCTCTGGATGACTATGTGAATACCCAGGGGGCTTCACTGTTCAGTGTCACTAAGAAG

CAGCTGGGAGCAGGAAGTATAGAAGAATGTGCAGCAAAATGTGAGGAGGACGAAGAATTC

ACCTGCAGGGCATTCCAATATCACAGTAAAGAGCAACAATGTGTGATAATGGCTGAAAAC

AGGAAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTTGAAAAGAAAGTGTAT

CTTCCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCCAAAACAAAA

AATGGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGACCTAGATTCTCACCT

GCTACACACCCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGACAACGATCCG

CAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGCGACATTCTT

GAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAG

ACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCACACGCTCATGGATAC

ATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGTCGTAACCCCGATAGG

GAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGGGAACTTTGCGACATC

CCCCGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAGTGTCTGAAGGGAACA

GGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCACACCTGTCAGCACTGG

AGTGCACAGACCCTCCACACACATAACAGGACACCAAAACTTCCCCTGCAAAAATTTG

GATGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGGTGCCATACAACCAAC

AGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCCTCCCCAGTATCCACG

GAACAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCAT

GGTGATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAGAAGTGTCAG

TCTTGGTCATCTATGACACCCACACCGGCACCAGAAGACCCCAGAAAACTACCCAAATGCT

GGCCTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGGTGTTTTACC

ACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCG

AGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAA

GACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGG

ACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAG

ACAAATCCACGGGCGGGTCTGGAAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGT

GGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAG

TGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGA

AGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGA

ACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACT

GCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCA

CACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTG

GAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGAC

AAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGT

TTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTTGGAGCTGGCCTTCTCAAGGAAGCC

CAGCTCCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTC

CAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGAC

AGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCT

TGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTT

GTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

SEQ ID NO: 66: Nucleic acid sequence of human lysine plasminogen gene

AAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCC

AAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGACCTAGA

TTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGAC

AACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGC

GACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAA

ATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCACACGCT

CATGGATACATTCCTTCCAAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGTCGTAAC

CCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCCAACAAGCGCTGGGAACTT

TGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAGTGTCTG

AAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCACACCTGT

CAGCACTGGAGTGCACAGACCCTCACACACACATAACAGGACACCAGAAAACTTCCCCTGC

AAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGGTGCCAT

ACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCCTCCCCA

GTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTCCAGGAC

TGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAG

AAGTGTCAGTCTTGGTCATCTATGACACCCACACCGGCACCAGAAGACCCCAGAAAACTAC

CCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGG

TGTTTTACCAGACCCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGGA

ACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCT

TCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACT

GTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTC

ACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAAATTACTGCCGTAACCCTGATGGT

GATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGAT

GTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAA

TGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTC

AGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGG

GTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATC

CTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGG

CTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTC

ATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGG

ACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTC

AAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAAT

GGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGC

CAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGA

GTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTT

TCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

references

(1) = Desire Collen, Thrombosis and Hemostasis

Haemostasis), 82, 1999

(2) ＝Forsgren et al., FEBS Lett.

213, 1987

(3) = Petersen et al., J.Biol.Chem., 265,

1990

(4) ＝Duman et al., Biotechnology and Applied Biochemistry (Biotechnol.Appl.

  Biochem.) 28; 39-45, 1998

(5) = Guan et al., Journal of Bioengineering (Sheng Wu Gong Cheng Xue Bao),

17, 2001

(6) = Gonzalez-Gronow et al., Biochimica et Biophysica Acta,

1039, 1990

(7) ＝Whitefleet-Smith et al., Biochemistry and Biophysics Literature (Arch.

Biochem.Biophys.), 271, 1989

(8) = Nilsen und Castellino, Protein expression and purification (Protein

Expression and Purification), 16, 1999

(9) ＝Busby et al., J.Biol.Chem., 266, 1991

(10) = Maniatis et al., Molecular Cloning: A Laboratory Manual (Molecular Cloning:

A Laboratory Manual), Cold Spring Harbor Press (Cold Spring Harbor

Press), 1989

(11) = Gassen & Schrimpf, Gentechnische Methoden, Spektrum

Akademischer Verlag, Heidelberg, 1999

(12) = Malinowski et al., Biochemistry (Biochemistry), 23, 1984

(13)=Stack et al., Biochem.J. 284, 1992

Sequence Listing

<110>N-Enzyme Biotechnology Co., Ltd.

<120> Method for producing recombinant protein in microorganism

<130>P04TK074108

<140>

<141>

<150>EP 02 002 716.5

<151>2002-02-06

<150>US60/357,809

<151>2002-02-21

<160>66

<170>PatentIn Ver.2.1

<210>1

<211>33

<212>DNA

<213> Homo sapiens

<400>1

aaaaaccgcg gtcaattatt tctcatcact ccc 33

<210>2

<211>37

<212>DNA

<213> Homo sapiens

<400>2

aaaaactcga gaaaagaaaa gtgtatctct cagagtg 37

<210>3

<211>49

<212>DNA

<213> Homo sapiens

<400>3

aaaaactcga gaaaagagag gctgaagcta aagtgtatct ctcagagtg 49

<210>4

<211>32

<212>DNA

<213> Homo sapiens

<400>4

aaaaattcga aaaatggaac ataaggaagt gg 32

<210>5

<211>35

<212>DNA

<213> Homo sapiens

<400>5

aaaaactcga gaaaagagag cctctggatg actat 35

<210>6

<211>47

<212>DNA

<213> Homo sapiens

<400>6

aaaaactcga gaaaagagag gctgaagctg agcctctgga tgactat 47

<210>7

<211>2400

<212>DNA

<213> Homo sapiens

<400>7

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagaaaagt gtatctctca gagtgcaaga ctgggaatgg aaagaactac 300

agagggacga tgtccaaaac aaaaaatggc atcacctgtc aaaaatggag ttccacttct 360

ccccacagac ctagattctc acctgctaca cacccctcag agggactgga ggagaactac 420

tgcaggaatc cagacaacga tccgcagggg ccctggtgct atactactga tccagaaaag 480

agatatgact actgcgacat tcttgagtgt gaagaggaat gtatgcattg cagtggagaa 540

aactatgacg gcaaaatttc caagaccatg tctggactgg aatgccaggc ctgggactct 600

cagagcccac acgctcatgg atacattcct tccaaatttc caaacaagaa cctgaagaag 660

aattactgtc gtaaccccga tagggagctg cggccttggt gtttcaccac cgaccccaac 720

aagcgctggg aactttgcga catcccccgc tgcacaacac ctccaccatc ttctggtccc 780

acctaccagt gtctgaaggg aacaggtgaa aactatcgcg ggaatgtggc tgttaccgtt 840

tccgggcaca cctgtcagca ctggagtgca cagacccctc acacacataa caggaccacca 900

gaaaacttcc cctgcaaaaa tttggatgaa aactactgcc gcaatcctga cggaaaagg 960

gccccatggt gccatacaac caacagccaa gtgcggtggg agtactgtaa gataccgtcc 1020

tgtgactcct ccccagtatc cacggaacaa ttggctccca cagcaccacc tgagctaacc 1080

cctgtggtcc aggactgcta ccatggtgat ggacagagct accgaggcac atcctccacc 1140

accaccacag gaaagaagtg tcagtcttgg tcatctatga caccacaccg gcaccagaag 1200

accccagaaa actacccaaa tgctggcctg acaatgaact actgcaggaa tccagatgcc 1260

gataaaggcc cctggtgttt taccacagac cccagcgtca ggtgggagta ctgcaacctg 1320

aaaaaatgct caggaacaga agcgagtgtt gtagcacctc cgcctgttgt cctgcttcca 1380

gatgtagaga ctccttccga agaagactgt atgtttggga atgggaaagg ataccgaggc 1440

aagagggcga ccactgttac tgggacgcca tgccaggact gggctgccca ggagccccat 1500

agacacagca ttttcactcc agagacaaat ccacgggcgg gtctggaaaa aaattactgc 1560

cgtaaccctg atggtgatgt aggtggtccc tggtgctaca cgacaaatcc aagaaaactt 1620

tacgactact gtgatgtccc tcagtgtgcg gccccttcat ttgattgtgg gaagcctcaa 1680

gtggagccga agaaatgtcc tggaagggtt gtgggggggt gtgtggccca cccacattcc 1740

tggccctggc aagtcagtct tagaacaagg tttggaatgc acttctgtgg aggcaccttg 1800

atatccccag agtgggtgtt gactgctgcc cactgcttgg agaagtcccc aaggccttca 1860

tcctacaagg tcatcctggg tgcacaccaa gaagtgaatc tcgaaccgca tgttcaggaa 1920

atagaagtgt ctaggctgtt cttggagccc acacgaaaag atattgcctt gctaaagcta 1980

agcagtcctg ccgtcatcac tgacaaagta atcccagctt gtctgccatc cccaaattat 2040

gtggtcgctg accggaccga atgtttcatc actggctggg gagaaaccca aggtactttt 2100

ggagctggcc ttctcaagga agcccagctc cctgtgattg agaataaagt gtgcaatcgc 2160

tatgagtttc tgaatggaag agtccaatcc accgaactct gtgctgggca tttggccgga 2220

ggcactgaca gttgccaggg tgacagtgga ggtcctctgg tttgcttcga gaaggacaaa 2280

tacattttac aaggagtcac ttcttggggt cttggctgtg cacgccccaa taagcctggt 2340

gtctatgttc gtgtttcaag gtttgttact tggattgagg gagtgatgag aaataattga 2400

<210>8

<211>799

<212>PRT

<213> Homo sapiens

<400>8

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn

85 90 95

Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr

100 105 110

Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro

115 120 125

Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro

130 135 140

Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys

145 150 155 160

Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu Glu Cys Met His

165 170 175

Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly

180 185 190

Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr

195 200 205

Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg

210 215 220

Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn

225 230 235 240

Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro

245 250 255

Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr

260 265 270

Arg Gly Asn Val Ala Val Thr Val Ser Gly His Thr Cys Gln His Trp

275 280 285

Ser Ala Gln Thr Pro His Thr His Asn Arg Thr Pro Glu Asn Phe Pro

290 295 300

Cys Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg

305 310 315 320

Ala Pro Trp Cys His Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys

325 330 335

Lys Ile Pro Ser Cys Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala

340 345 350

Pro Thr Ala Pro Pro Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His

355 360 365

Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly

370 375 380

Lys Lys Cys Gln Ser Trp Ser Ser Ser Met Thr Pro His Arg His Gln Lys

385 390 395 400

Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg

405 410 415

Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser

420 425 430

Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala

435 440 445

Ser Val Val Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr

450 455 460

Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly

465 470 475 480

Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala

485 490 495

Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg

500 505 510

Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly

515 520 525

Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys

530 535 540

Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln

545 550 555 560

Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala

565 570 575

His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly

580 585 590

Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr

595 600 605

Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val

610 615 620

Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu

625 630 635 640

Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala

645 650 655

Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro

660 665 670

Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys

675 680 685

Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu

690 695 700

Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg

705 710 715 720

Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly

725 730 735

His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro

740 745 750

Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser

755 760 765

Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg

770 775 780

Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

785 790 795

<210>9

<211>2412

<212>DNA

<213> Homo sapiens

<400>9

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagaggc tgaagctaaa gtgtatctct cagagtgcaa gactgggaat 300

ggaaagaact acagagggac gatgtccaaa acaaaaaatg gcatcacctg tcaaaaatgg 360

agttccactt ctccccacag acctagattc tcacctgcta cacacccctc agagggactg 420

gaggagaact actgcaggaa tccagacaac gatccgcagg ggccctggtg ctatactact 480

gatccagaaa agagatatga ctactgcgac attcttgagt gtgaagagga atgtatgcat 540

tgcagtggag aaaactatga cggcaaaatt tccaagacca tgtctggact ggaatgccag 600

gcctgggact ctcagagccc acacgctcat ggatacattc cttccaaatt tccaaacaag 660

aacctgaaga agaattactg tcgtaaccccc gataggagc tgcggccttg gtgtttcacc 720

accgacccca acaagcgctg ggaactttgc gacatccccc gctgcacaac acctccacca 780

tcttctggtc ccacctacca gtgtctgaag ggaacaggtg aaaactatcg cgggaatgtg 840

gctgttaccg tttccgggca cacctgtcag cactggagtg cacagacccc tcacacacat 900

aacaggacac cagaaaactt cccctgcaaa aatttggatg aaaactactg ccgcaatcct 960

gacggaaaaa gggccccatg gtgccataca accaacagcc aagtgcggtg ggagtactgt 1020

aagataccgt cctgtgactc ctccccagta tccacggaac aattggctcc cacagcacca 1080

cctgagctaa cccctgtggt ccaggactgc taccatggtg atggacagag ctaccgaggc 1140

acatcctcca ccaccaccac aggaaagaag tgtcagtctt ggtcatctat gacaccacac 1200

cggcaccaga agaccccaga aaactaccca aatgctggcc tgacaatgaa ctactgcagg 1260

aatccagatg ccgataaagg cccctggtgt tttaccacag accccagcgt caggtgggag 1320

tactgcaacc tgaaaaaatg ctcaggaaca gaagcgagtg ttgtagcacc tccgcctgtt 1380

gtcctgcttc cagatgtaga gactccttcc gaagaagact gtatgtttgg gaatgggaaa 1440

ggataccgag gcaagagggc gaccactgtt actgggacgc catgccagga ctgggctgcc 1500

caggagcccc atagacacag cattttcact ccagagacaa atccacgggc gggtctggaa 1560

aaaaattact gccgtaaccc tgatggtgat gtaggtggtc cctggtgcta cacgacaaat 1620

ccaagaaaac tttacgacta ctgtgatgtc cctcagtgtg cggccccttc atttgattgt 1680

gggaagcctc aagtggagcc gaagaaatgt cctggaaggg ttgtgggggg gtgtgtggcc 1740

cacccacatt cctggccctg gcaagtcagt cttagaacaa ggtttggaat gcacttctgt 1800

ggaggcacct tgatatcccc agagtgggtg ttgactgctg cccactgctt ggagaagtcc 1860

ccaaggcctt catcctacaa ggtcatcctg ggtgcacacc aagaagtgaa tctcgaaccg 1920

catgttcagg aaatagaagt gtctaggctg ttcttggagc ccacacgaaa agatattgcc 1980

ttgctaaagc taagcagtcc tgccgtcatc actgacaaag taatcccagc ttgtctgcca 2040

tccccaaatt atgtggtcgc tgaccggacc gaatgtttca tcactggctg gggagaaacc 2100

caaggtactt ttggagctgg ccttctcaag gaagcccagc tccctgtgat tgagaataaa 2160

gtgtgcaatc gctatgagtt tctgaatgga agagtccaat ccaccgaact ctgtgctggg 2220

catttggccg gaggcactga cagttgccag ggtgacagtg gaggtcctct ggtttgcttc 2280

gagaaggaca aatacatttt acaaggagtc acttcttggg gtcttggctg tgcacgcccc 2340

aataagcctg gtgtctatgt tcgtgtttca aggtttgtta cttggattga gggagtgatg 2400

agaaata ga 2412

<210>10

<211>803

<212>PRT

<213> Homo sapiens

<400>10

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala Lys Val Tyr Leu Ser Glu Cys

85 90 95

Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr Lys

100 105 110

Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg Pro

115 120 125

Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn Tyr

130 135 140

Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr Thr

145 150 155 160

Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu Glu

165 170 175

Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser Lys

180 185 190

Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His

195 200 205

Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys

210 215 220

Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe Thr

225 230 235 240

Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys Thr

245 250 255

Thr Pro Pro Pro Ser Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly Thr

260 265 270

Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val Ser Gly His Thr

275 280 285

Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His Asn Arg Thr Pro

290 295 300

Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn Pro

305 310 315 320

Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn Ser Gln Val Arg

325 330 335

Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser Pro Val Ser Thr

340 345 350

Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr Pro Val Val Gln

355 360 365

Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser Ser Thr

370 375 380

Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser Met Thr Pro His

385 390 395 400

Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr Met

405 410 415

Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe Thr

420 425 430

Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser

435 440 445

Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val Val Leu Leu Pro

450 455 460

Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys

465 470 475 480

Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln

485 490 495

Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu

500 505 510

Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp

515 520 525

Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu

530 535 540

Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys

545 550 555 560

Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly

565 570 575

Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg

580 585 590

Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu

595 600 605

Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser

610 615 620

Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro

625 630 635 640

His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg

645 650 655

Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp

660 665 670

Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp

675 680 685

Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe

690 695 700

Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys

705 710 715 720

Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu

725 730 735

Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp

740 745 750

Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln

755 760 765

Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly

770 775 780

Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met

785 790 795 800

Arg Asn Asn

<210>11

<211>2433

<212>DNA

<213> Homo sapiens

<400>11

atggaacata aggaagtggt tcttctactt cttttatttc tgaaatcagg tcaaggagag 60

cctctggatg actatgtgaa tacccagggg gcttcactgt tcagtgtcac taagaagcag 120

ctgggagcag gaagtataga agaatgtgca gcaaaatgtg aggaggacga agaattcacc 180

tgcagggcat tccaatatca cagtaaagag caacaatgtg tgataatggc tgaaaacagg 240

aagtcctcca taatcattag gatgagagat gtagttttat ttgaaaagaa agtgtatctc 300

tcagagtgca agactgggaa tggaaagaac tacagaggga cgatgtccaa aacaaaaaat 360

ggcatcacct gtcaaaaatg gagttccact tctccccaca gacctagatt ctcacctgct 420

acacacccct cagagggact ggaggagaac tactgcagga atccagacaa cgatccgcag 480

gggccctggt gctatactac tgatccagaa aagagattg actactgcga cattcttgag 540

tgtgaagagg aatgtatgca ttgcagtgga gaaaactatg acggcaaaat ttccaagacc 600

atgtctggac tggaatgcca ggcctgggac tctcagagcc cacacgctca tggatacatt 660

ccttccaaat ttccaaacaa gaacctgaag aagaattact gtcgtaaccc cgatagggag 720

ctgcggcctt ggtgtttcac caccgacccc aacaagcgct gggaactttg cgacatcccc 780

cgctgcacaa cacctccacc atcttctggt cccacctacc agtgtctgaa gggaacaggt 840

gaaaactatc gcgggaatgt ggctgttacc gtttccgggc acacctgtca gcactggagt 900

gcacagaccc ctcacacaca taacaggaca ccagaaaact tcccctgcaa aaatttggat 960

gaaaactact gccgcaatcc tgacggaaaa agggccccat ggtgccatac aaccaacagc 1020

caagtgcggt gggagtactg taagataccg tcctgtgact cctccccagt atccacggaa 1080

caattggctc ccacagcacc acctgagcta acccctgtgg tccaggactg ctaccatggt 1140

gatggacaga gctaccgagg cacatcctcc accacccaca caggaaagaa gtgtcagtct 1200

tggtcatcta tgacaccaca ccggcaccag aagaccccag aaaactaccc aaatgctggc 1260

ctgacaatga actactgcag gaatccagat gccgataaag gcccctggtg ttttaccaca 1320

gaccccagcg tcaggtggga gtactgcaac ctgaaaaaat gctcaggaac agaagcgagt 1380

gttgtagcac ctccgcctgt tgtcctgctt ccagatgtag agactccttc cgaagaagac 1440

tgtatgtttg ggaatgggaa aggataccga ggcaagaggg cgaccactgt tactgggacg 1500

ccatgccagg actgggctgc ccaggagccc catagacaca gcattttcac tccagagaca 1560

aatccacggg cgggtctgga aaaaaattac tgccgtaacc ctgatggtga tgtaggtggt 1620

ccctggtgct acacgacaaa tccaagaaaa ctttacgact actgtgatgt ccctcagtgt 1680

gcggcccctt catttgattg tgggaagcct caagtggagc cgaagaaatg tcctggaagg 1740

gttgtggggg ggtgtgtggc ccaccacat tcctggccct ggcaagtcag tcttagaaca 1800

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 1860

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 1920

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 1980

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 2040

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 2100

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 2160

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 2220

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 2280

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 2340

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 2400

acttggattg agggagtgat gagaaataat tga 2433

<210>12

<211>810

<212>PRT

<213> Homo sapiens

<400>12

Met Glu His Lys Glu Val Val Leu Leu Leu Leu Leu Phe Leu Lys Ser

1 5 10 15

Gly Gln Gly Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser

20 25 30

Leu Phe Ser Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu

35 40 45

Cys Ala Ala Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe

50 55 60

Gln Tyr His Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg

65 70 75 80

Lys Ser Ser Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys

85 90 95

Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg

100 105 110

Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser

115 120 125

Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser

130 135 140

Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln

145 150 155 160

Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys

165 170 175

Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn

180 185 190

Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala

195 200 205

Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe

210 215 220

Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu

225 230 235 240

Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu

245 250 255

Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr

260 265 270

Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala

275 280 285

Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro

290 295 300

His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp

305 310 315 320

Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His

325 330 335

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys

340 345 350

Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro

355 360 365

Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser

370 375 380

Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser

385 390 395 400

Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr

405 410 415

Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp

420 425 430

Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr

435 440 445

Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro

450 455 460

Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp

465 470 475 480

Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr

485 490 495

Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg

500 505 510

His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys

515 520 525

Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr

530 535 540

Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys

545 550 555 560

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys

565 570 575

Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp

580 585 590

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly

595 600 605

Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu

610 615 620

Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His

625 630 635 640

Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg

645 650 655

Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser

660 665 670

Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser

675 680 685

Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp

690 695 700

Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln

705 710 715 720

Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn

725 730 735

Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly

740 745 750

Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu

755 760 765

Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys

770 775 780

Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val

785 790 795 800

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

805 810

<210>13

<211>2631

<212>DNA

<213> Homo sapiens

<400>13

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagagcc tctggatgac tatgtgaata cccaggggggc ttcactgttc 300

agtgtcacta agaagcagct gggagcagga agtatagaag aatgtgcagc aaaatgtgag 360

gaggacgaag aattcacctg cagggcattc caatatcaca gtaaagagca acaatgtgtg 420

ataatggctg aaaacaggaa gtcctccata atcattagga tgagagatgt agttttattt 480

gaaaagaaag tgtatctctc agagtgcaag actgggaatg gaaagaacta cagagggacg 540

atgtccaaaa caaaaaatgg catcacctgt caaaaatgga gttccacttc tccccacaga 600

cctagattct cacctgctac acacccctca gagggactgg aggagaacta ctgcaggaat 660

ccagacaacg atccgcaggg gccctggtgc tatactactg atccagaaaa gagatatgac 720

tactgcgaca ttcttgagtg tgaagaggaa tgtatgcatt gcagtggaga aaactatgac 780

ggcaaaattt ccaagaccat gtctggactg gaatgccagg cctgggactc tcagagccca 840

cacgctcatg gatacattcc ttccaaattt ccaaacaaga acctgaagaa gaattactgt 900

cgtaaccccg atagggagct gcggccttgg tgtttcacca ccgaccccaa caagcgctgg 960

gaactttgcg acatcccccg ctgcacaaca cctccaccat cttctggtcc cacctaccag 1020

tgtctgaagg gaacaggtga aaactatcgc gggaatgtgg ctgttaccgt ttccgggcac 1080

acctgtcagc actggagtgc acagaccct cacacacata acaggaccc agaaaacttc 1140

ccctgcaaaa atttggatga aaactactgc cgcaatcctg acggaaaaag ggccccatgg 1200

tgccatacaa ccaacagcca agtgcggtgg gagtactgta agataccgtc ctgtgactcc 1260

tccccagtat ccacggaaca attggctccc acagcaccac ctgagctaac ccctgtggtc 1320

caggactgct accatggtga tggacagagc taccgaggca catcctccac caccaccaca 1380

ggaaagaagt gtcagtcttg gtcatctatg acaccacacc ggcaccagaa gaccccagaa 1440

aactacccaa atgctggcct gacaatgaac tactgcagga atccagatgc cgataaaggc 1500

ccctggtgtt ttaccacaga ccccagcgtc aggtgggagt actgcaacct gaaaaaatgc 1560

tcaggaacag aagcgagtgt tgtagcacct ccgcctgttg tcctgcttcc agatgtagag 1620

actccttccg aagaagactg tatgtttggg aatgggaaag gataccgagg caagagggcg 1680

accactgtta ctgggacgcc atgccaggac tgggctgccc aggagcccca tagacacagc 1740

attttcactc cagagacaaa tccacgggcg ggtctggaaa aaaattactg ccgtaaccct 1800

gatggtgatg taggtggtcc ctggtgctac acgacaaatc caagaaaact ttacgactac 1860

tgtgatgtcc ctcagtgtgc ggccccttca tttgattgtg ggaagcctca agtggagccg 1920

aagaaatgtc ctggaagggt tgtggggggg tgtgtggccc accccacattc ctggccctgg 1980

caagtcagtc ttagaacaag gtttggaatg cacttctgtg gaggcacctt gatatcccca 2040

gagtgggtgt tgactgctgc ccactgcttg gagaagtccc caaggccttc atcctacaag 2100

gtcatcctgg gtgcacacca agaagtgaat ctcgaaccgc atgttcagga aatagaagtg 2160

tctaggctgt tcttggagcc cacacgaaaa gatattgcct tgctaaagct aagcagtcct 2220

gccgtcatca ctgacaaagt aatcccagct tgtctgccat ccccaaatta tgtggtcgct 2280

gaccggaccg aatgtttcat cactggctgg ggagaaaccc aaggtacttt tggagctggc 2340

cttctcaagg aagcccagct ccctgtgatt gagaataaag tgtgcaatcg ctatgagttt 2400

ctgaatggaa gagtccaatc caccgaactc tgtgctgggc atttggccgg aggcactgac 2460

agttgccagg gtgacagtgg aggtcctctg gtttgcttcg agaaggacaa atacatttta 2520

caaggagtca cttcttgggg tcttggctgt gcacgcccca ataagcctgg tgtctatgtt 2580

cgtgtttcaa ggtttgttac ttggattgag ggagtgatga gaaataattg a 2631

<210>14

<211>876

<212>PRT

<213> Homo sapiens

<400>14

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly

85 90 95

Ala Ser Leu Phe Ser Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile

100 105 110

Glu Glu Cys Ala Ala Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg

115 120 125

Ala Phe Gln Tyr His Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu

130 135 140

Asn Arg Lys Ser Ser Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe

145 150 155 160

Glu Lys Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn

165 170 175

Tyr Arg Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys

180 185 190

Trp Ser Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His

195 200 205

Pro Ser Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp

210 215 220

Pro Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp

225 230 235 240

Tyr Cys Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly

245 250 255

Glu Asn Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys

260 265 270

Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser

275 280 285

Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp

290 295 300

Arg Glu Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp

305 310 315 320

Glu Leu Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly

325 330 335

Pro Thr Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn

340 345 350

Val Ala Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln

355 360 365

Thr Pro His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn

370 375 380

Leu Asp Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp

385 390 395 400

Cys His Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro

405 410 415

Ser Cys Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala

420 425 430

Pro Pro Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly

435 440 445

Gln Ser Tyr Arg Gly Thr Ser Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys

450 455 460

Gln Ser Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu

465 470 475 480

Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp

485 490 495

Ala Asp Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp

500 505 510

Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val

515 520 525

Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu

530 535 540

Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala

545 550 555 560

Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro

565 570 575

His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu

580 585 590

Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp

595 600 605

Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro

610 615 620

Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro

625 630 635 640

Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His

645 650 655

Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe

660 665 670

Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His

675 680 685

Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly

690 695 700

Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val

705 710 715 720

Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys

725 730 735

Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu

740 745 750

Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr

755 760 765

Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu

770 775 780

Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe

785 790 795 800

Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala

805 810 815

Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys

820 825 830

Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu

835 840 845

Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg

850 855 860

Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

865 870 875

<210>15

<211>2643

<212>DNA

<213> Homo sapiens

<400>15

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagaggc tgaagctgag cctctggatg actatgtgaa tacccagggg 300

gcttcactgt tcagtgtcac taagaagcag ctgggagcag gaagtataga agaatgtgca 360

gcaaaatgtg aggaggacga agaattcacc tgcagggcat tccaatatca cagtaaagag 420

caacaatgtg tgataatggc tgaaaacagg aagtcctcca taatcattag gatgagagat 480

gtagttttat ttgaaaagaa agtgtatctc tcagagtgca agactgggaa tggaaagaac 540

tacagaggga cgatgtccaa aacaaaaaat ggcatcacct gtcaaaaatg gagttccact 600

tctccccaca gacctagatt ctcacctgct acacacccct cagagggact ggaggagaac 660

tactgcagga atccagacaa cgatccgcag gggccctggt gctatactac tgatccagaa 720

aagagatatg actactgcga cattcttgag tgtgaagagg aatgtatgca ttgcagtgga 780

gaaaactatg acggcaaaat ttccaagacc atgtctggac tggaatgcca ggcctgggac 840

tctcagagcc cacacgctca tggatacatt ccttccaaat ttccaaacaa gaacctgaag 900

aagaattact gtcgtaaccc cgatagggag ctgcggcctt ggtgtttcac caccgacccc 960

aacaagcgct gggaactttg cgacatcccc cgctgcacaa cacctccacc atcttctggt 1020

cccacctacc agtgtctgaa gggaacaggt gaaaactatc gcgggaatgt ggctgttacc 1080

gtttccgggc acacctgtca gcactggagt gcacagaccc ctcacacaca taacaggaca 1140

ccagaaaact tcccctgcaa aaatttggat gaaaactact gccgcaatcc tgacggaaaa 1200

agggccccat ggtgccatac aaccaacagc caagtgcggt gggagtactg taagataccg 1260

tcctgtgact cctccccagt atccacggaa caattggctc ccacagcacc acctgagcta 1320

acccctgtgg tccaggactg ctaccatggt gatggacaga gctaccgagg cacatcctcc 1380

accaccca caggaaagaa gtgtcagtct tggtcatcta tgacaccaca ccggcaccag 1440

aagaccccag aaaactaccc aaatgctggc ctgacaatga actactgcag gaatccagat 1500

gccgataaag gcccctggtg ttttaccaca gaccccagcg tcaggtggga gtactgcaac 1560

ctgaaaaaat gctcaggaac agaagcgagt gttgtagcac ctccgcctgt tgtcctgctt 1620

ccagatgtag agactccttc cgaagaagac tgtatgtttg ggaatgggaa aggataccga 1680

ggcaagaggg cgaccactgt tactgggacg ccatgccagg actgggctgc ccaggagccc 1740

catagacaca gcattttcac tccagagaca aatccacggg cgggtctgga aaaaaattac 1800

tgccgtaacc ctgatggtga tgtaggtggt ccctggtgct acacgacaaa tccaagaaaa 1860

ctttacgact actgtgatgt ccctcagtgt gcggcccctt catttgattg tgggaagcct 1920

caagtggagc cgaagaaatg tcctggaagg gttgtggggg ggtgtgtggc ccaccacat 1980

tcctggccct ggcaagtcag tcttagaaca aggtttggaa tgcacttctg tggaggcacc 2040

ttgatatccc cagagtgggt gttgactgct gcccactgct tggagaagtc cccaaggcct 2100

tcatcctaca aggtcatcct gggtgcacac caagaagtga atctcgaacc gcatgttcag 2160

gaaatagaag tgtctaggct gttcttggag cccacacgaa aagatattgc cttgctaaag 2220

ctaagcagtc ctgccgtcat cactgacaaa gtaatcccag cttgtctgcc atccccaaat 2280

tatgtggtcg ctgaccggac cgaatgtttc atcactggct ggggagaaac ccaaggtact 2340

tttggagctg gccttctcaa ggaagcccag ctccctgtga ttgagaataa agtgtgcaat 2400

cgctatgagt ttctgaatgg aagagtccaa tccaccgaac tctgtgctgg gcatttggcc 2460

ggaggcactg acagttgcca gggtgacagt gaggtcctc tggtttgctt cgagaaggac 2520

aaatacattt tacaaggagt cacttcttgg ggtcttggct gtgcacgccc caataagcct 2580

ggtgtctatg ttcgtgtttc aaggtttgtt acttggattg agggagtgat gagaaataat 2640

tga 2643

<210>16

<211>880

<212>PRT

<213> Homo sapiens

<400>16

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Ash Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala Glu Pro Leu Asp Asp Tyr Val

85 90 95

Asn Thr Gln Gly Ala Ser Leu Phe Ser Val Thr Lys Lys Gln Leu Gly

100 105 110

Ala Gly Ser Ile Glu Glu Cys Ala Ala Lys Cys Glu Glu Asp Glu Glu

115 120 125

Phe Thr Cys Arg Ala Phe Gln Tyr His Ser Lys Glu Gln Gln Cys Val

130 135 140

Ile Met Ala Glu Asn Arg Lys Ser Ser Ile Ile Ile Arg Met Arg Asp

145 150 155 160

Val Val Leu Phe Glu Lys Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly

165 170 175

Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr Lys Asn Gly Ile

180 185 190

Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg Pro Arg Phe Ser

195 200 205

Pro Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn

210 215 220

Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu

225 230 235 240

Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu Glu Glu Cys Met

245 250 255

His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser

260 265 270

Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly

275 280 285

Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys

290 295 300

Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro

305 310 315 320

Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro

325 330 335

Pro Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn

340 345 350

Tyr Arg Gly Asn Val Ala Val Thr Val Ser Gly His Thr Cys Gln His

355 360 365

Trp Ser Ala Gln Thr Pro His Thr His Asn Arg Thr Pro Glu Asn Phe

370 375 380

Pro Cys Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys

385 390 395 400

Arg Ala Pro Trp Cys His Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr

405 410 415

Cys Lys Ile Pro Ser Cys Asp Ser Ser Ser Pro Val Ser Thr Glu Gln Leu

420 425 430

Ala Pro Thr Ala Pro Pro Glu Leu Thr Pro Val Val Gln Asp Cys Tyr

435 440 445

His Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Thr

450 455 460

Gly Lys Lys Cys Gln Ser Trp Ser Ser Met Thr Pro His Arg His Gln

465 470 475 480

Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys

485 490 495

Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro

500 505 510

Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu

515 520 525

Ala Ser Val Val Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu

530 535 540

Thr Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg

545 550 555 560

Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala

565 570 575

Ala Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro

580 585 590

Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val

595 600 605

Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr

610 615 620

Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro

625 630 635 640

Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val

645 650 655

Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe

660 665 670

Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu

675 680 685

Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys

690 695 700

Val Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln

705 710 715 720

Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile

725 730 735

Ala Leu Leu Lys Leu Ser Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile

740 745 750

Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu

755 760 765

Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly

770 775 780

Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn

785 790 795 800

Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala

805 810 815

Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly

820 825 830

Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr

835 840 845

Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val

850 855 860

Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

865 870 875 880

<210>17

<211>791

<212>PRT

<213> Homo sapiens

<400>17

Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser

1 5 10 15

Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala

20 25 30

Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His

35 40 45

Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser

50 55 60

Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr

65 70 75 80

Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met

85 90 95

Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser

100 105 110

Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu

115 120 125

Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp

130 135 140

Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu

145 150 155 160

Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly

165 170 175

Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser

180 185 190

Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys

195 200 205

Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro

210 215 220

Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile

225 230 235 240

Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys

245 250 255

Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val

260 265 270

Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His

275 280 285

Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr

290 295 300

Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn

305 310 315 320

Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser

325 330 335

Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr

340 345 350

Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly

355 360 365

Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser

370 375 380

Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala

385 390 395 400

Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro

405 410 415

Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu

420 425 430

Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val

435 440 445

Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe

450 455 460

Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly

465 470 475 480

Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile

485 490 495

Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys

500 505 510

Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn

515 520 525

Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro

530 535 540

Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly

545 550 555 560

Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln

565 570 575

Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu

580 585 590

Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser

595 600 605

Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val

610 615 620

Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu

625 630 635 640

Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala

645 650 655

Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr

660 665 670

Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr

675 680 685

Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val

690 695 700

Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val

705 710 715 720

Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser

725 730 735

Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys

740 745 750

Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro

755 760 765

Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile

770 775 780

Glu Gly Val Met Arg Asn Asn

785 790

<210>18

<211>714

<212>PRT

<213> Homo sapiens

<400>18

Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg

1 5 10 15

Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser

20 25 30

Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser

35 40 45

Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln

50 55 60

Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys

65 70 75 80

Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn

85 90 95

Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala

100 105 110

Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe

115 120 125

Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu

130 135 140

Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu

145 150 155 160

Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr

165 170 175

Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala

180 185 190

Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro

195 200 205

His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp

210 215 220

Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His

225 230 235 240

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys

245 250 255

Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro

260 265 270

Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser

275 280 285

Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser

290 295 300

Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr

305 310 315 320

Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp

325 330 335

Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr

340 345 350

Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro

355 360 365

Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp

370 375 380

Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr

385 390 395 400

Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg

405 410 415

His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys

420 425 430

Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr

435 440 445

Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys

450 455 460

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys

465 470 475 480

Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp

485 490 495

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly

500 505 510

Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu

515 520 525

Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His

530 535 540

Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg

545 550 555 560

Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser

565 570 575

Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser

580 585 590

Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp

595 600 605

Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln

610 615 620

Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn

625 630 635 640

Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly

645 650 655

Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu

660 665 670

Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys

675 680 685

Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val

690 695 700

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

705 710

<210>19

<211>33

<212>DNA

<213> Homo sapiens

<400>19

aaaaactcga gaaaagagca cctccgcctg ttg 33

<210>20

<211>45

<212>DNA

<213> Homo sapiens

<400>20

aaaaactcga gaaaagagag gctgaagctg cacctccgcc tgttg 45

<210>2l

<211>34

<212>DNA

<213> Homo sapiens

<400>21

aaaaactcga gaaaagaaaa ctttacgact actg 34

<210>22

<211>46

<212>DNA

<213> Homo sapiens

<400>22

aaaaactcga gaaaagagag gctgaagcta aactttacga ctactg 46

<210>23

<211>33

<212>DNA

<213> Homo sapiens

<400>23

aaaaactcga gaaaagactt tacgactact gtg 33

<210>24

<211>45

<212>DNA

<213> Homo sapiens

<400>24

aaaaactcga gaaaagagag gctgaagctc tttacgacta ctgtg 45

<210>25

<211>36

<212>DNA

<213> Homo sapiens

<400>25

aaaaactcga gaaaagagcc ccttcatttg attgtg 36

<210>26

<211>48

<212>DNA

<213> Homo sapiens

<400>26

aaaaactcga gaaaagagag gctgaagctg ccccttcatt tgattgtg 48

<210>27

<211>37

<212>DNA

<213> Homo sapiens

<400>27

aaaaactcga gaaaagatca tttgattgtg ggaagcc 37

<210>28

<21l>49

<212>DNA

<213> Homo sapiens

<400>28

aaaaactcga gaaaagagag gctgaagctt catttgattg tgggaagcc 49

<210>29

<21l>348

<212>PRT

<213> Homo sapiens

<400>29

Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu

1 5 10 15

Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala

20 25 30

Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro

35 40 45

His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu

50 55 60

Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp

65 70 75 80

Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro

85 90 95

Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro

100 105 110

Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His

115 120 125

Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe

130 135 140

Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His

145 150 155 160

Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly

165 170 175

Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val

180 185 190

Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys

195 200 205

Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu

210 215 220

Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr

225 230 235 240

Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu

245 250 255

Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe

260 265 270

Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala

275 280 285

Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys

290 295 300

Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu

305 310 315 320

Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg

325 330 335

Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

340 345

<210>30

<211>261

<212>PRT

<213> Homo sapiens

<400>30

Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe

1 5 10 15

Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val

20 25 30

Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser

35 40 45

Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser

50 55 60

Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg

65 70 75 80

Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn Leu

85 90 95

Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro

100 105 110

Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile

115 120 125

Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val

130 135 140

Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly

145 150 155 160

Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu

165 170 175

Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser

180 185 190

Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln

195 200 205

Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile

210 215 220

Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys

225 230 235 240

Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly

245 250 255

Val Met Arg Asn Asn

260

<210>31

<211>260

<212>PRT

<213> Homo sapiens

<400>31

Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp

1 5 10 15

Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val

20 25 30

Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu

35 40 45

Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro

50 55 60

Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro

65 70 75 80

Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu

85 90 95

Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr

100 105 110

Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr

115 120 125

Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala

130 135 140

Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr

145 150 155 160

Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn

165 170 175

Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr

180 185 190

Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly

195 200 205

Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu

210 215 220

Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro

225 230 235 240

Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val

245 250 255

Met Arg Asn Asn

260

<210>32

<211>249

<212>PRT

<213> Homo sapiens

<400>32

Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys

1 5 10 15

Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro

20 25 30

Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly

35 40 45

Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu

50 55 60

Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln

65 70 75 80

Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu

85 90 95

Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser

100 105 110

Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro

115 120 125

Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly

130 135 140

Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu

145 150 155 160

Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly

165 170 175

Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr

180 185 190

Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys

195 200 205

Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala

210 215 220

Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr

225 230 235 240

Trp Ile Glu Gly Val Met Arg Asn Asn

245

<210>33

<211>247

<212>PRT

<213> Homo sapiens

<400>33

Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly

1 5 10 15

Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln

20 25 30

Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu

35 40 45

Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser

50 55 60

Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val

65 70 75 80

Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu

85 90 95

Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala

100 105 110

Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr

115 120 125

Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr

130 135 140

Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val

145 150 155 160

Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val

165 170 175

Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser

180 185 190

Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys

195 200 205

Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro

210 215 220

Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile

225 230 235 240

Glu Gly Val Met Arg Asn Asn

245

<210>34

<211>249

<212>DNA

<213> Homo sapiens

<400>34

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgag 249

<210>35

<211>83

<212>PRT

<213> Homo sapiens

<400>35

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu

<210>36

<211>6

<212>DNA

<213> Homo sapiens

<400>36

aaaaga 6

<210>37

<211>12

<212>DNA

<213> Homo sapiens

<400>37

gaggctgaag ct 12

<210>38

<211>2

<212>PRT

<213> Homo sapiens

<400>38

Lys Arg

l

<210>39

<211>4

<212>PRT

<213> Homo sapiens

<400>39

Glu Ala Glu Ala

1

<210>40

<211>433

<212>PRT

<213> Homo sapiens

<400>40

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val

85 90 95

Glu Thr Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr

100 105 110

Arg Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp

115 120 125

Ala Ala Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu Thr Asn

130 135 140

Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp

145 150 155 160

Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp

165 170 175

Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys

180 185 190

Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys

195 200 205

Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg

210 215 220

Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val

225 230 235 240

Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr

245 250 255

Lys Val Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val

260 265 270

Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp

275 280 285

Ile Ala Leu Leu Lys Leu Ser Ser Ser Pro Ala Val Ile Thr Asp Lys Val

290 295 300

Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr

305 310 315 320

Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala

325 330 335

Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys

340 345 350

Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys

355 360 365

Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly

370 375 380

Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val

385 390 395 400

Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr

405 410 415

Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn

420 425 430

Asn

<210>41

<211>437

<212>PRT

<213> Homo sapiens

<400>41

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala Ala Pro Pro Pro Pro Val Val Leu

85 90 95

Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe Gly Asn

100 105 110

Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro

115 120 125

Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile Phe Thr

130 135 140

Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn

145 150 155 160

Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg

165 170 175

Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe

180 185 190

Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val

195 200 205

Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser

210 215 220

Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser

225 230 235 240

Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg

245 250 255

Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn Leu

260 265 270

Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro

275 280 285

Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile

290 295 300

Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val

305 310 315 320

Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly

325 330 335

Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu

340 345 350

Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser

355 360 365

Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln

370 375 380

Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile

385 390 395 400

Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys

405 410 415

Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly

420 425 430

Val Met Arg Asn Asn

435

<210>42

<211>346

<212>PRT

<213> Homo sapiens

<400>42

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys

85 90 95

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys

100 105 110

Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp

115 120 125

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly

130 135 140

Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu

145 150 155 160

Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His

165 170 175

Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg

180 185 190

Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser

195 200 205

Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser

210 215 220

Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp

225 230 235 240

Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln

245 250 255

Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn

260 265 270

Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly

275 280 285

Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu

290 295 300

Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys

305 310 315 320

Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val

325 330 335

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

340 345

<210>43

<211>350

<212>PRT

<213> Homo sapiens

<400>43

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala Lys Leu Tyr Asp Tyr Cys Asp

85 90 95

Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val

100 105 110

Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His

115 120 125

Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met

130 135 140

His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala

145 150 155 160

Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile

165 170 175

Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile

180 185 190

Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu

195 200 205

Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala

210 215 220

Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe

225 230 235 240

Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu

245 250 255

Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr

260 265 270

Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His

275 280 285

Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu

290 295 300

Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp

305 310 315 320

Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val

325 330 335

Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

340 345 350

<210>44

<211>345

<212>PRT

<213> Homo sapiens

<400>44

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala

85 90 95

Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys

100 105 110

Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro

115 120 125

Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly

130 135 140

Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu

145 150 155 160

Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln

165 170 175

Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu

180 185 190

Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser

195 200 205

Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro

210 215 220

Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly

225 230 235 240

Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu

245 250 255

Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly

260 265 270

Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr

275 280 285

Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys

290 295 300

Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala

305 310 315 320

Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr

325 330 335

Trp Ile Glu Gly Val Met Arg Asn Asn

340 345

<210>45

<211>349

<212>PRT

<213> Homo sapiens

<400>45

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala Leu Tyr Asp Tyr Cys Asp Val

85 90 95

Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu

100 105 110

Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro

115 120 125

His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His

130 135 140

Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala

145 150 155 160

His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu

165 170 175

Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu

180 185 190

Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu

195 200 205

Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys

210 215 220

Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile

225 230 235 240

Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys

245 250 255

Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu

260 265 270

Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu

275 280 285

Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val

290 295 300

Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly

305 310 315 320

Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser

325 330 335

Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

340 345

<210>46

<211>334

<212>PRT

<213> Homo sapiens

<400>46

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val

85 90 95

Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His

100 105 110

Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met

115 120 125

His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala

130 135 140

Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile

145 150 155 160

Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile

165 170 175

Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu

180 185 190

Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala

195 200 205

Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe

210 215 220

Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu

225 230 235 240

Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr

245 250 255

Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His

260 265 270

Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu

275 280 285

Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp

290 295 300

Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val

305 310 315 320

Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

325 330

<210>47

<211>338

<212>PRT

<213> Homo sapiens

<400>47

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala Ala Pro Ser Phe Asp Cys Gly

85 90 95

Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly

100 105 110

Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr

115 120 125

Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp

130 135 140

Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser

145 150 155 160

Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His

165 170 175

Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys

180 185 190

Asp Ile Ala Leu Leu Lys Leu Ser Ser Ser Pro Ala Val Ile Thr Asp Lys

195 200 205

Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg

210 215 220

Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly

225 230 235 240

Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val

245 250 255

Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu

260 265 270

Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser

275 280 285

Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly

290 295 300

Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val

305 310 315 320

Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg

325 330 335

Asn Asn

<210>48

<21l>332

<212>PRT

<213> Homo sapiens

<400>48

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro

85 90 95

Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His

100 105 110

Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe

115 120 125

Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His

130 135 140

Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly

145 150 155 160

Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val

165 170 175

Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys

180 185 190

Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu

195 200 205

Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr

210 215 220

Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu

225 230 235 240

Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe

245 250 255

Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala

260 265 270

Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys

275 280 285

Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu

290 295 300

Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg

305 310 315 320

Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

325 330

<210>49

<211>336

<212>PRT

<213> Homo sapiens

<400>49

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala Ser Phe Asp Cys Gly Lys Pro

85 90 95

Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val

100 105 110

Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe

115 120 125

Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu

130 135 140

Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys

145 150 155 160

Val Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln

165 170 175

Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile

180 185 190

Ala Leu Leu Lys Leu Ser Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile

195 200 205

Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu

210 215 220

Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly

225 230 235 240

Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn

245 250 255

Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala

260 265 270

Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly

275 280 285

Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr

290 295 300

Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val

305 310 315 320

Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

325 330 335

<210>50

<211>1302

<212>DNA

<213> Homo sapiens

<400>50

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagcacc tccgcctgtt gtcctgcttc cagatgtaga gactccttcc 300

gaagaagact gtatgtttgg gaatgggaaa ggataccgag gcaagagggc gaccactgtt 360

actgggacgc catgccagga ctgggctgcc caggagcccc atagacacag cattttcact 420

ccagagacaa atccacgggc gggtctggaa aaaaattact gccgtaaccc tgatggtgat 480

gtaggtggtc cctggtgcta cacgacaaat ccaagaaaac tttacgacta ctgtgatgtc 540

cctcagtgtg cggccccttc atttgattgt gggaagcctc aagtggagcc gaagaaatgt 600

cctggaaggg ttgtgggggg gtgtgtggcc cacccacatt cctggccctg gcaagtcagt 660

cttagaacaa ggtttggaat gcacttctgt ggaggcacct tgatatcccc agagtgggtg 720

ttgactgctg cccactgctt ggagaagtcc ccaaggcctt catcctacaa ggtcatcctg 780

ggtgcacacc aagaagtgaa tctcgaaccg catgttcagg aaatagaagt gtctaggctg 840

ttcttggagc ccacacgaaa agatattgcc ttgctaaagc taagcagtcc tgccgtcatc 900

actgacaaag taatcccagc ttgtctgcca tccccaaatt atgtggtcgc tgaccggacc 960

gaatgtttca tcactggctg gggagaaacc caaggtactt ttggagctgg ccttctcaag 1020

gaagcccagc tccctgtgat tgagaataaa gtgtgcaatc gctatgagtt tctgaatgga 1080

agagtccaat ccaccgaact ctgtgctggg catttggccg gaggcactga cagttgccag 1140

ggtgacagtg gaggtcctct ggtttgcttc gagaaggaca aatacatttt acaaggagtc 1200

acttcttggg gtcttggctg tgcacgcccc aataagcctg gtgtctatgt tcgtgtttca 1260

aggtttgtta cttggattga gggagtgatg agaaataatt ga 1302

<210>51

<211>1314

<212>DNA

<213> Homo sapiens

<400>51

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagaggc tgaagctgca cctccgcctg ttgtcctgct tccagatgta 300

gagactcctt ccgaagaaga ctgtatgttt gggaatggga aaggataccg aggcaagagg 360

gcgaccactg ttactgggac gccatgccag gactgggctg cccaggagcc ccatagacac 420

agcattttca ctccagagac aaatccacgg gcgggtctgg aaaaaaatta ctgccgtaac 480

cctgatggtg atgtaggtgg tccctggtgc tacacgacaa atccaagaaa actttacgac 540

tactgtgatg tccctcagtg tgcggcccct tcatttgatt gtgggaagcc tcaagtggag 600

ccgaagaaat gtcctggaag ggttgtgggg gggtgtgtgg cccacccaca ttcctggccc 660

tggcaagtca gtcttagaac aaggtttgga atgcacttct gtggaggcac cttgatatcc 720

ccagagtggg tgttgactgc tgcccactgc ttggagaagt ccccaaggcc ttcatcctac 780

aaggtcatcc tgggtgcaca ccaagaagtg aatctcgaac cgcatgttca ggaaatagaa 840

gtgtctaggc tgttcttgga gccccacacga aaagatattg ccttgctaaa gctaagcagt 900

cctgccgtca tcactgacaa agtaatccca gcttgtctgc catccccaaa ttatgtggtc 960

gctgaccgga ccgaatgttt catcactggc tggggagaaa cccaaggtac ttttggagct 1020

ggccttctca aggaagccca gctccctgtg attgagaata aagtgtgcaa tcgctatgag 1080

tttctgaatg gaagagtcca atccaccgaa ctctgtgctg ggcatttggc cggaggcact 1140

gacagttgcc agggtgacag tggaggtcct ctggtttgct tcgagaagga caaatacatt 1200

ttacaaggag tcacttcttg gggtcttggc tgtgcacgcc ccaataagcc tggtgtctat 1260

gttcgtgttt caaggtttgt tacttggatt gagggagtga tgagaaataa ttga 1314

<210>52

<211>1041

<212>DNA

<213> Homo sapiens

<400>52

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagaaaact ttacgactac tgtgatgtcc ctcagtgtgc ggccccttca 300

tttgattgtg ggaagcctca agtggagccg aagaaatgtc ctggaagggt tgtggggggg 360

tgtgtggccc accccacattc ctggccctgg caagtcagtc ttagaacaag gtttggaatg 420

cacttctgtg gaggcacctt gatatcccca gagtgggtgt tgactgctgc ccactgcttg 480

gagaagtccc caaggccttc atcctacaag gtcatcctgg gtgcacacca agaagtgaat 540

ctcgaaccgc atgttcagga aatagaagtg tctaggctgt tcttggagcc cacacgaaaa 600

gatattgcct tgctaaagct aagcagtcct gccgtcatca ctgacaaagt aatcccagct 660

tgtctgccat ccccaaatta tgtggtcgct gaccggaccg aatgtttcat cactggctgg 720

ggagaaaccc aaggtacttt tggagctggc cttctcaagg aagcccagct ccctgtgatt 780

gagaataaag tgtgcaatcg ctatgagttt ctgaatggaa gagtccaatc caccgaactc 840

tgtgctgggc atttggccgg aggcactgac agttgccagg gtgacagtgg aggtcctctg 900

gtttgcttcg agaaggacaa atacatttta caaggagtca cttcttgggg tcttggctgt 960

gcacgcccca ataagcctgg tgtctatgtt cgtgtttcaa ggtttgttac ttggattgag 1020

ggagtgatga gaaataattg a 1041

<210>53

<211>1053

<212>DNA

<213> Homo sapiens

<400>53

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagaggc tgaagctaaa ctttacgact actgtgatgt ccctcagtgt 300

gcggcccctt catttgattg tgggaagcct caagtggagc cgaagaaatg tcctggaagg 360

gttgtggggg ggtgtgtggc ccaccacat tcctggccct ggcaagtcag tcttagaaca 420

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 480

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 540

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 600

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 660

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 720

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 780

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 840

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 900

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 960

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 1020

acttggattg agggagtgat gagaaataat tga 1053

<210>54

<211>1038

<212>DNA

<213> Homo sapiens

<400>54

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagacttta cgactactgt gatgtccctc agtgtgcggc cccttcattt 300

gattgtggga agcctcaagt ggagccgaag aaatgtcctg gaagggttgt gggggggtgt 360

gtggcccacc cacattcctg gccctggcaa gtcagtctta gaacaaggtt tggaatgcac 420

ttctgtggag gcaccttgat atccccagag tgggtgttga ctgctgccca ctgcttggag 480

aagtccccaa ggccttcatc ctacaaggtc atcctgggtg cacaccaaga agtgaatctc 540

gaaccgcatg ttcaggaaat agaagtgtct aggctgttct tggagcccac acgaaaagat 600

attgccttgc taaagctaag cagtcctgcc gtcatcactg acaaagtaat cccagcttgt 660

ctgccatccc caaattatgt ggtcgctgac cggaccgaat gtttcatcac tggctgggga 720

gaaacccaag gtacttttgg agctggcctt ctcaaggaag cccagctccc tgtgattgag 780

aataaagtgt gcaatcgcta tgagtttctg aatggaagag tccaatccac cgaactctgt 840

gctgggcatt tggccggagg cactgacagt tgccagggtg acagtgggagg tcctctggtt 900

tgcttcgaga aggacaaata cattttacaa gagtcactt cttggggtct tggctgtgca 960

cgccccaata agcctggtgt ctatgttcgt gtttcaaggt ttgttacttg gattgaggga 1020

gtgatgagaa ataattga 1038

<210>55

<211>1050

<212>DNA

<213> Homo sapiens

<400>55

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagaggc tgaagctctt tacgactact gtgatgtccc tcagtgtgcg 300

gccccttcat ttgattgtgg gaagcctcaa gtggagccga agaaatgtcc tggaagggtt 360

gtgggggggt gtgtggccca cccacattcc tggccctggc aagtcagtct tagaacaagg 420

tttggaatgc acttctgtgg aggcaccttg atatccccag agtgggtgtt gactgctgcc 480

cactgcttgg agaagtcccc aaggccttca tcctacaagg tcatcctggg tgcacaccaa 540

gaagtgaatc tcgaaccgca tgttcaggaa atagaagtgt ctaggctgtt cttggagccc 600

acacgaaaag atattgcctt gctaaagcta agcagtcctg ccgtcatcac tgacaaagta 660

atcccagctt gtctgccatc cccaaattat gtggtcgctg accggaccga atgtttcatc 720

actggctggg gagaaaccca aggtactttt ggagctggcc ttctcaagga agcccagctc 780

cctgtgattg agaataaagt gtgcaatcgc tatgagtttc tgaatggaag agtccaatcc 840

accgaactct gtgctgggca tttggccgga ggcactgaca gttgccaggg tgacagtgga 900

ggtcctctgg tttgcttcga gaaggacaaa tacattttac aaggagtcac ttcttggggt 960

cttggctgtg cacgccccaa taagcctggt gtctatgttc gtgtttcaag gtttgttact 1020

tggattgagg gagtgatgag aaataattga 1050

<210>56

<211>1005

<212>DNA

<213> Homo sapiens

<400>56

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 300

tgtcctggaa gggttgtggg ggggtgtgtg gccccaccac attcctggcc ctggcaagtc 360

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 420

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 480

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 540

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 600

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 660

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 720

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 780

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 840

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 900

gtcacttctt gggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 960

tcaaggtttg ttacttggat tgagggagtg atgagaaata attga 1005

<210>57

<211>1017

<212>DNA

<213> Homo sapiens

<400>57

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagaggc tgaagctgcc ccttcatttg attgtgggaa gcctcaagtg 300

gagccgaaga aatgtcctgg aagggttgtg ggggggtgtg tggcccaccc attcctgg 360

ccctggcaag tcagtcttag aacaaggttt ggaatgcact tctgtggagg caccttgata 420

tccccagagt gggtgttgac tgctgcccac tgcttggaga agtccccaag gccttcatcc 480

tacaaggtca tcctgggtgc acaccaagaa gtgaatctcg aaccgcatgt tcaggaaata 540

gaagtgtcta ggctgttctt ggagcccaca cgaaaagata ttgccttgct aaagctaagc 600

agtcctgccg tcatcactga caaagtaatc ccagcttgtc tgccatcccc aaattatgtg 660

gtcgctgacc ggaccgaatg tttcatcact ggctggggag aaacccaagg tacttttgga 720

gctggccttc tcaaggaagc ccagctccct gtgattgaga ataaagtgtg caatcgctat 780

gagtttctga atggaagagt ccaatccacc gaactctgtg ctgggcattt ggccggaggc 840

actgacagtt gccagggtga cagtggaggt cctctggttt gcttcgagaa ggacaaatac 900

attttacaag gagtcacttc ttggggtctt ggctgtgcac gccccaataa gcctggtgtc 960

tatgttcgtg tttcaaggtt tgttacttgg attgagggag tgatgagaaa taattga 1017

<210>58

<211>999

<212>DNA

<213> Homo sapiens

<400>58

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagatcatt tgattgtggg aagcctcaag tggagccgaa gaaatgtcct 300

ggaagggttg tgggggggtg tgtggcccac ccacattcct ggccctggca agtcagtctt 360

agaacaaggt ttggaatgca cttctgtgga ggcaccttga tatccccaga gtgggtgttg 420

actgctgccc actgcttgga gaagtcccca aggccttcat cctacaaggt catcctgggt 480

gcacaccaag aagtgaatct cgaaccgcat gttcaggaaa tagaagtgtc taggctgttc 540

ttggagccca cacgaaaaga tattgccttg ctaaagctaa gcagtcctgc cgtcatcact 600

gacaaagtaa tcccagcttg tctgccatcc ccaaattatg tggtcgctga ccggaccgaa 660

tgtttcatca ctggctgggg agaaacccaa ggtacttttg gagctggcct tctcaaggaa 720

gcccagctcc ctgtgattga gaataaagtg tgcaatcgct atgagtttct gaatggaaga 780

gtccaatcca ccgaactctg tgctgggcat ttggccggag gcactgacag ttgccagggt 840

gacagtggag gtcctctggt ttgcttcgag aaggacaaat aattttaca aggagtcact 900

tcttggggtc ttggctgtgc acgccccaat aagcctggtg tctatgttcg tgtttcaagg 960

tttgttactt ggattgaggg agtgatgaga aataattga 999

<210>59

<211>1011

<212>DNA

<213> Homo sapiens

<400>59

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagaggc tgaagcttca tttgattgtg ggaagcctca agtggagccg 300

aagaaatgtc ctggaagggt tgtggggggg tgtgtggccc accccacattc ctggccctgg 360

caagtcagtc ttagaacaag gtttggaatg cacttctgtg gaggcacctt gatatcccca 420

gagtgggtgt tgactgctgc ccactgcttg gagaagtccc caaggccttc atcctacaag 480

gtcatcctgg gtgcacacca agaagtgaat ctcgaaccgc atgttcagga aatagaagtg 540

tctaggctgt tcttggagcc cacacgaaaa gatattgcct tgctaaagct aagcagtcct 600

gccgtcatca ctgacaaagt aatcccagct tgtctgccat ccccaaatta tgtggtcgct 660

gaccggaccg aatgtttcat cactggctgg ggagaaaccc aaggtacttt tggagctggc 720

cttctcaagg aagcccagct ccctgtgatt gagaataaag tgtgcaatcg ctatgagttt 780

ctgaatggaa gagtccaatc caccgaactc tgtgctgggc atttggccgg aggcactgac 840

agttgccagg gtgacagtgg aggtcctctg gtttgcttcg agaaggacaa atacatttta 900

caaggagtca cttcttgggg tcttggctgt gcacgcccca ataagcctgg tgtctatgtt 960

cgtgtttcaa ggtttgttac ttggattgag ggagtgatga gaaataattg a 1011

<210>60

<211>1047

<212>DNA

<213> Homo sapiens

<400>60

gcacctccgc ctgttgtcct gcttccagat gtagagactc cttccgaaga agactgtatg 60

tttgggaatg ggaaaggata ccgaggcaag agggcgacca ctgttactgg gacgccatgc 120

caggactggg ctgcccagga gccccataga cacagcattt tcactccaga gacaaatcca 180

cgggcgggtc tggaaaaaaa ttactgccgt aaccctgatg gtgatgtagg tggtccctgg 240

tgctacacga caaatccaag aaaactttac gactactgtg atgtccctca gtgtgcggcc 300

ccttcatttg attgtgggaa gcctcaagtg gagccgaaga aatgtcctgg aagggttgtg 360

ggggggtgtg tggcccaccc aattcctgg ccctggcaag tcagtcttag aacaaggttt 420

ggaatgcact tctgtggagg caccttgata tccccagagt gggtgttgac tgctgcccac 480

tgcttggaga agtccccaag gccttcatcc tacaaggtca tcctgggtgc acaccaagaa 540

gtgaatctcg aaccgcatgt tcaggaaata gaagtgtcta ggctgttctt ggagcccaca 600

cgaaaagata ttgccttgct aaagctaagc agtcctgccg tcatcactga caaagtaatc 660

ccagcttgtc tgccatcccc aaattatgtg gtcgctgacc ggaccgaatg tttcatcact 720

ggctggggag aaacccaagg tacttttgga gctggccttc tcaaggaagc ccagctccct 780

gtgattgaga ataaagtgtg caatcgctat gagtttctga atggaagagt ccaatccacc 840

gaactctgtg ctgggcattt ggccggaggc actgacagtt gccagggtga cagtggaggt 900

cctctggttt gcttcgagaa ggacaaatac attttacaag gagtcacttc ttggggtctt 960

ggctgtgcac gccccaataa gcctggtgtc tatgttcgtg tttcaaggtt tgttacttgg 1020

attgagggag tgatgagaaa taattga 1047

<210>61

<211>786

<212>DNA

<213> Homo sapiens

<400>61

aaactttacg actactgtga tgtccctcag tgtgcggccc cttcatttga ttgtgggaag 60

cctcaagtgg agccgaagaa atgtcctgga agggttgtgg gggggtgtgt ggccaccca 120

cattcctggc cctggcaagt cagtcttaga acaaggtttg gaatgcactt ctgtggaggc 180

accttgatat ccccagagtg ggtgttgact gctgcccact gcttggagaa gtccccaagg 240

ccttcatcct acaaggtcat cctgggtgca caccaagaag tgaatctcga accgcatgtt 300

caggaaatag aagtgtctag gctgttcttg gagccccacac gaaaagatat tgccttgcta 360

aagctaagca gtcctgccgt catcactgac aaagtaatcc cagcttgtct gccatcccca 420

aattatgtgg tcgctgaccg gaccgaatgt ttcatcactg gctggggaga aacccaaggt 480

acttttggag ctggccttct caaggaagcc cagctccctg tgattgagaa taaagtgtgc 540

aatcgctatg agtttctgaa tggaagagtc caatccaccg aactctgtgc tgggcatttg 600

gccggaggca ctgacagttg ccagggtgac agtggaggtc ctctggtttg cttcgagaag 660

gacaaataca ttttacaagg agtcacttct tggggtcttg gctgtgcacg ccccaataag 720

cctggtgtct atgttcgtgt ttcaaggttt gttacttgga ttgagggagt gatgagaaat 780

aattga 786

<210>62

<211>783

<212>DNA

<213> Homo sapiens

<400>62

ctttacgact actgtgatgt ccctcagtgt gcggccccctt catttgattg tgggaagcct 60

caagtggagc cgaagaaatg tcctggaagg gttgtggggg ggtgtgtggc ccaccacat 120

tcctggccct ggcaagtcag tcttagaaca aggtttggaa tgcacttctg tggaggcacc 180

ttgatatccc cagagtgggt gttgactgct gcccactgct tggagaagtc cccaaggcct 240

tcatcctaca aggtcatcct gggtgcacac caagaagtga atctcgaacc gcatgttcag 300

gaaatagaag tgtctaggct gttcttggag cccacacgaa aagatattgc cttgctaaag 360

ctaagcagtc ctgccgtcat cactgacaaa gtaatcccag cttgtctgcc atccccaaat 420

tatgtggtcg ctgaccggac cgaatgtttc atcactggct ggggagaaac ccaaggtact 480

tttggagctg gccttctcaa ggaagcccag ctccctgtga ttgagaataa agtgtgcaat 540

cgctatgagt ttctgaatgg aagagtccaa tccaccgaac tctgtgctgg gcatttggcc 600

ggaggcactg acagttgcca gggtgacagt gaggtcctc tggtttgctt cgagaaggac 660

aaatacattt tacaaggagt cacttcttgg ggtcttggct gtgcacgccc caataagcct 720

ggtgtctatg ttcgtgtttc aaggtttgtt acttggattg agggagtgat gagaaataat 780

tga 783

<210>63

<211>750

<212>DNA

<213> Homo sapiens

<400>63

gccccttcat ttgattgtgg gaagcctcaa gtggagccga agaaatgtcc tggaagggtt 60

gtgggggggt gtgtggccca cccacattcc tggccctggc aagtcagtct tagaacaagg 120

tttggaatgc acttctgtgg aggcaccttg atatccccag agtgggtgtt gactgctgcc 180

cactgcttgg agaagtcccc aaggccttca tcctacaagg tcatcctggg tgcacaccaa 240

gaagtgaatc tcgaaccgca tgttcaggaa atagaagtgt ctaggctgtt cttggagccc 300

acacgaaaag atattgcctt gctaaagcta agcagtcctg ccgtcatcac tgacaaagta 360

atcccagctt gtctgccatc cccaaattat gtggtcgctg accggaccga atgtttcatc 420

actggctggg gagaaaccca aggtactttt ggagctggcc ttctcaagga agcccagctc 480

cctgtgattg agaataaagt gtgcaatcgc tatgagtttc tgaatggaag agtccaatcc 540

accgaactct gtgctgggca tttggccgga ggcactgaca gttgccaggg tgacagtgga 600

ggtcctctgg tttgcttcga gaaggacaaa tacattttac aaggagtcac ttcttggggt 660

cttggctgtg cacgccccaa taagcctggt gtctatgttc gtgtttcaag gtttgttact 720

tggattgagg gagtgatgag aaataattga 750

<210>64

<211>744

<212>DNA

<213> Homo sapiens

<400>64

tcatttgatt gtgggaagcc tcaagtggag ccgaagaaat gtcctggaag ggttgtgggg 60

gggtgtgtgg cccacccaca ttcctggccc tggcaagtca gtcttagaac aaggtttgga 120

atgcacttct gtggaggcac cttgatatcc ccagagtggg tgttgactgc tgcccactgc 180

ttggagaagt ccccaaggcc ttcatcctac aaggtcatcc tgggtgcaca ccaagaagtg 240

aatctcgaac cgcatgttca ggaaatagaa gtgtctaggc tgttcttgga gccccacacga 300

aaagatattg ccttgctaaa gctaagcagt cctgccgtca tcactgacaa agtaatccca 360

gcttgtctgc catccccaaa ttatgtggtc gctgaccgga ccgaatgttt catcactggc 420

tggggagaaa cccaaggtac ttttggagct ggccttctca aggaagccca gctccctgtg 480

attgagaata aagtgtgcaa tcgctatgag tttctgaatg gaagagtcca atccaccgaa 540

ctctgtgctg ggcatttggc cggaggcact gacagttgcc agggtgacag tggaggtcct 600

ctggtttgct tcgagaagga caaatacatt ttacaaggag tcacttcttg gggtcttggc 660

tgtgcacgcc ccaataagcc tggtgtctat gttcgtgttt caaggtttgt tacttggatt 720

gagggagtga tgagaaataa ttga 744

<210>65

<211>2376

<212>DNA

<213> Homo sapiens

<400>65

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctggggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480

gagtgtgaag aggaatgtat gcattgcagt ggagaaaact atgacggcaa aatttccaag 540

accatgtctg gactggaatg ccaggcctgg gactctcaga gcccacacgc tcatggatac 600

attccttcca aatttccaaa caagaacctg aagaagaatt actgtcgtaa ccccgatagg 660

gagctgcggc cttggtgttt caccaccgac cccaacaagc gctgggaact ttgcgacatc 720

ccccgctgca caacacctcc accatcttct ggtcccacct accagtgtct gaagggaaca 780

ggtgaaaact atcgcgggaa tgtggctgtt accgtttccg ggcaacacctg tcagcactgg 840

agtgcacaga cccctcacac acataacagg acaccagaaa acttcccctg caaaaatttg 900

gatgaaaact actgccgcaa tcctgacgga aaaagggccc catggtgcca tacaaccaac 960

agccaagtgc ggtgggagta ctgtaagata ccgtcctgtg actcctcccc agtatccacg 1020

gaacaattgg ctcccacagc accacctgag ctaacccctg tggtccagga ctgctaccat 1080

ggtgatggac agagctaccg aggcacatcc tccaccacca ccacaggaaa gaagtgtcag 1140

tcttggtcat ctatgacacc acaccggcac cagaagaccc cagaaaacta cccaaatgct 1200

ggcctgacaa tgaactactg caggaatcca gatgccgata aaggcccctg gtgttttacc 1260

acagacccca gcgtcaggtg ggagtactgc aacctgaaaa aatgctcagg aacagaagcg 1320

agtgttgtag cacctccgcc tgttgtcctg cttccagatg tagagactcc ttccgaagaa 1380

gactgtatgt ttgggaatgg gaaaggatac cgaggcaaga gggcgaccac tgttactggg 1440

acgccatgcc aggactgggc tgcccaggag ccccatagac acagcatttt cactccagag 1500

acaaatccac gggcgggtct ggaaaaaaat tactgccgta accctgatgg tgatgtaggt 1560

ggtccctggt gctacacgac aaatccaaga aaactttacg actactgtga tgtccctcag 1620

tgtgcggccc cttcatttga ttgtgggaag cctcaagtgg agccgaagaa atgtcctgga 1680

agggttgtgg gggggtgtgt ggcccaccca cattcctggc cctggcaagt cagtcttaga 1740

acaaggtttg gaatgcactt ctgtggaggc accttgatat ccccagagtg ggtgttgact 1800

gctgcccact gcttggagaa gtccccaagg ccttcatcct acaaggtcat cctgggtgca 1860

caccaagaag tgaatctcga accgcatgtt caggaaatag aagtgtctag gctgttcttg 1920

gagcccacac gaaaagatat tgccttgcta aagctaagca gtcctgccgt catcactgac 1980

aaagtaatcc cagcttgtct gccatcccca aattatgtgg tcgctgaccg gaccgaatgt 2040

ttcatcactg gctggggaga aacccaaggt acttttggag ctggccttct caaggaagcc 2100

cagctccctg tgattgagaa taaagtgtgc aatcgctatg agtttctgaa tggaagagtc 2160

caatccaccg aactctgtgc tgggcatttg gccggaggca ctgacagttg ccagggtgac 2220

agtggaggtc ctctggtttg cttcgagaag gacaaataca ttttacaagg agtcacttct 2280

tggggtcttg gctgtgcacg ccccaataag cctggtgtct atgttcgtgt ttcaaggttt 2340

gttacttgga ttgagggagt gatgagaaat aattga 2376

<210>66

<211>2145

<212>DNA

<213> Homo sapiens

<400>66

aaagtgtatc tctcagagtg caagactggg aatggaaaga actacagagg gacgatgtcc 60

aaaacaaaaa atggcatcac ctgtcaaaaa tggagttcca cttctcccca cagacctaga 120

ttctcacctg ctacacaccc ctcagaggga ctggaggaga actactgcag gaatccagac 180

aacgatccgc aggggccctg gtgctatact actgatccag aaaagagata tgactactgc 240

gacattcttg agtgtgaaga ggaatgtatg cattgcagtg gagaaaacta tgacggcaaa 300

atttccaaga ccatgtctgg actggaatgc caggcctggg actctcagag cccacacgct 360

catggataca ttccttccaa atttccaaac aagaacctga agaagaatta ctgtcgtaac 420

cccgataggg agctgcggcc ttggtgtttc accaccgacc ccaacaagcg ctgggaactt 480

tgcgacatcc cccgctgcac aacacctcca ccatcttctg gtcccaccta ccagtgtctg 540

aagggaacag gtgaaaacta tcgcgggaat gtggctgtta ccgtttccgg gcacacctgt 600

cagcactgga gtgcacagac ccctcacaca cataacagga caccagaaaa cttcccctgc 660

aaaaatttgg atgaaaacta ctgccgcaat cctgacggaa aaagggcccc atggtgccat 720

acaaccaaca gccaagtgcg gtgggagtac tgtaagatac cgtcctgtga ctcctcccca 780

gtatccacgg aacaattggc tcccacagca ccacctgagc taacccctgt ggtccaggac 840

tgctaccatg gtgatggaca gagctaccga ggcacatcct ccaccaccac cacaggaaag 900

aagtgtcagt cttggtcatc tatgacacca caccggcacc agaagacccc agaaaactac 960

ccaaatgctg gcctgacaat gaactactgc aggaatccag atgccgataa aggcccctgg 1020

tgttttacca cagaccccag cgtcaggtgg gagtactgca acctgaaaaa atgctcagga 1080

acagaagcga gtgttgtagc acctccgcct gttgtcctgc ttccagatgt agagactcct 1140

tccgaagaag actgtatgtt tgggaatggg aaaggatacc gaggcaagag ggcgaccact 1200

gttactggga cgccatgcca ggactgggct gcccaggagc cccatagaca cagcattttc 1260

actccagaga caaatccacg ggcgggtctg gaaaaaaatt actgccgtaa ccctgatggt 1320

gatgtaggtg gtccctggtg ctacacgaca aatccaagaa aactttacga ctactgtgat 1380

gtccctcagt gtgcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 1440

tgtcctggaa gggttgtggg ggggtgtgtg gccccaccac attcctggcc ctggcaagtc 1500

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 1560

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 1620

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 1680

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 1740

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 1800

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 1860

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1920

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1980

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 2040

gtcacttctt gggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 2100

tcaaggtttg ttacttggat tgagggagtg atgagaaata attga 2145

Claims (62)

1. A method for recombinantly producing functional plasminogen in microorganisms, said method at least comprising the following steps: a) fusion of a nucleic acid sequence encoding at least a functional part of plasminogen with a nucleic acid sequence encoding at least one segment of a signal peptide, in order to cleave the signal peptide, said nucleic acid sequence encoding functional plasminogen and at least a nucleic acid encoding a signal peptide The sequences are coupled by protease cleavage site codons. 2. The recombinant production method according to claim 1, wherein said nucleic acid sequence encoding at least a functional part of plasminogen comprises a proteolytic domain of plasminogen, or a variant or fragment thereof. 3. The recombinant production method according to claim 1 or 2, wherein said nucleic acid sequence encoding at least a functional part of plasminogen encodes glutamate plasminogen or lysine plasminogen. 4. The recombinant production method according to any one of claims 1 to 3, wherein the nucleic acid sequence encoding at least one signal peptide encodes a pre-propeptide, propeptide or propeptide; and/or the codon encodes a protease Restriction sites for Kex2 and/or Ste13. 5. The recombinant production method according to claim 4, wherein the nucleic acid sequence encoding at least one signal peptide encodes the signal peptide of α factor, or the signal peptide of SUC2, PHA-E or PHO1 of Saccharomyces cerevisiae. 6. The recombinant production method according to any one of the preceding claims, characterized in that at least 20 mg/l of functional glutamate plasminogen or lysine plasminogen is produced. 7. The recombinant production method according to any one of the preceding claims, characterized in that at least 120 U/l of functional lysine plasminogen is produced after 120 hours of treatment. 8. The recombinant production method according to any one of the preceding claims, wherein the two oligonucleotide primers used for amplification are selected from the group consisting of: NO36a (Seq. ID No.19), NO36b (Seq. ID No. 20), NO36c(Seq.ID No.21), NO36d(Seq.ID No.22), NO36e(Seq.ID No.23), NO36f(Seq.ID No.24), NO36g(Seq.ID No.24) 25), NO36h (Seq. ID No. 26), NO36i (Seq. ID No. 27) or NO36j (Seq. ID No. 28). 9. The recombinant production method according to any one of the preceding claims, wherein the two oligonucleotide primers used for amplification are selected from the group consisting of: NO34 (Seq. ID No.1), NO36 (Seq. ID No. 2), NO57(Seq.ID No.3), NO37(Seq.ID No.4), NO35(Seq.ID No.5), NO56(Seq.ID No.6); and selected from: NO36a(Seq.ID No.4); .ID No.19), NO36b(Seq.ID No.20), NO36c(Seq.ID No.21), NO36d(Seq.ID No.22), NO36e(Seq.ID No.23), NO36f(Seq.ID No.23) ID No. 24), NO36g (Seq. ID No. 25), NO36h (Seq. ID No. 26), NO36i (Seq. ID No. 27) or NO36j (Seq. ID No. 28). 10. The recombinant production method according to any one of the preceding claims, wherein the fusion product produced in step a) is integrated into an expression vector suitable for microorganisms. 11. The recombinant production method according to claim 10, wherein said expression vector is suitable for fungi. 12. The recombinant production method according to claim 10 or 11, wherein the expression vector comprises an inducible promoter or a constitutive promoter. 13. The recombinant production method according to claim 10 or 11, wherein said expression vector comprises a constitutive GAP promoter of Pichia pastoris. 14. The recombinant production method according to claim 12, wherein the obtained nucleic acid is a plasmid, preferably the plasmid is selected from the group consisting of: pPLG11.2, pPLG12.1, pPLG13.1, pPLG14.2, pPLG15.1 , pPLG16.3, pPLG17.2, pPLG18.1, pPLG19.2, pPLG20.1, pAC37.1, pJW9.1, pMHS476.1, pSM54.2, pSM49.8, pSM82.1 or pSM58.1. 15. The recombinant production method according to any one of the preceding claims, characterized in that, the codon encodes the enzyme cleavage site of protease Kex2; the fusion gene of plasminogen has the following nucleic acid sequence: Seq. ID No. 7, 13, 50, 52, 54, 56 or 58. 16. The recombinant production method according to any one of the preceding claims, characterized in that, the codon encodes the enzyme cleavage site of protease Kex2; the fusion protein of plasminogen has the following amino acid sequence: Seq. ID No. 8, 14, 40, 42, 44, 46 or 48. 17. The recombinant production method according to any one of the preceding claims, characterized in that, the codons encode the enzyme cleavage sites of proteases Kex2 and Ste13; the fusion gene of plasminogen has the following nucleic acid Sequence: Seq. ID No. 9, 15, 51, 53, 55 or 57. 18. The recombinant production method according to any one of the preceding claims, characterized in that, the codons encode the enzyme cleavage sites of proteases Kex2 and Ste13; the fusion protein of plasminogen has the following amino acid sequence: Seq .ID No. 10, 16, 41, 43, 45, 47 or 49. 19. The recombinant production method according to any one of the preceding claims, wherein the microbial host is transformed with the plasmid according to the method of claim 14. 20. The recombinant production method according to any one of the preceding claims, wherein the host used is a eukaryotic microorganism. 21. The recombinant production method according to claim 20, characterized in that the host used belongs to the genus Fungi. 22. The recombinant production method according to claim 21, wherein the host used belongs to the genus Pichia, Saccharomyces, Hansenula, Candida or Aspergillus. 23. The recombinant production method according to claim 22, wherein the host used belongs to the following species: Pichia pastoris, Pichia methanolica, Hansenula polymorpha (Hansenula polymorpha), Aspergillus niger, Aspergillus oryzae or Aspergillus nidulans. 24. The recombinant production method according to any one of the preceding claims, wherein the overexpressed nucleic acid sequence encodes at least a functional part of plasminogen, and said at least functional part of plasminogen is secreted by a host microorganism, The host microorganism is transformed from the fusion product produced in step a). 25. The recombinant production method according to any one of the preceding claims, wherein the functional part of the plasminogen nucleic acid sequence is one of the following sequences: Seq. ID No. 60, 61, 62, 63, 64, 65 or 66. 26. The recombinant production method according to any one of the preceding claims, characterized in that a functional human plasminogen is produced. 27. The recombinant production method according to any one of the preceding claims, characterized in that a functional plasminogen homologous to human is produced. 28. A plasmid, which is plasmid pPLG11.2, plasmid pPLG12.1, plasmid pPLG13.1, plasmid pPLG14.2, plasmid pPLG15.1, plasmid pPLG16.3, plasmid pPLG17.2, plasmid pPLG18.1, plasmid pPLG19 .2 or plasmid pPLG20.1. 29. A plasmid which is plasmid pMHS476.1, plasmid pSM54.2, plasmid pSM49.8 or plasmid pSM82.1. 30. A plasmid which is plasmid pSM58.1, plasmid pAC37.1 or plasmid pJW9.1. 31. The nucleic acid sequence obtained by the recombinant production method according to any one of claims 1 to 27, characterized in that at least the nucleic acid sequence encoding the functional part of plasminogen is operable with a promoter active in yeast Coupling; the nucleic acid sequence encoding at least the functional part of plasminogen is also coupled with at least one nucleic acid sequence encoding a signal peptide, the nucleic acid sequence encoding functional plasminogen and at least the nucleic acid sequence encoding signal peptide are composed of Codon coupling of the cleavage sites of proteases Kex2 and Ste13. 32. Plasminogen obtained by the recombinant production method according to any one of claims 1-27. 33. The plasminogen according to claim 32, characterized in that, the plasminogen is microplasminogen, small plasminogen, lysine plasminogen, glutamic acid fibrinolytic Enzyme or plasminogen derivatives. 34. The plasminogen derivative according to claim 33, characterized in that the derivative comprises a functional proteolytic domain of plasminogen and comprises at least one deletion, and/or at least one amino acid substitution, And/or fused to at least another amino acid or at least a stretch of peptide or at least one protein. 35. The plasminogen derivative according to claim 33 or 34, characterized in that said derivative is activated into active plasmin by at least one plasminogen activator. 36. The plasminogen derivative according to any one of claims 33 to 35, characterized in that said derivative comprises at least a functional proteolytic domain of plasminogen and is combined with microplasminogen, The homology of small plasminogen, lysine plasminogen or glutamate plasminogen is higher than 80%, preferably higher than 90%, particularly preferably higher than 95%. 37. Plasmin obtained by activating plasminogen or a plasminogen derivative according to any one of claims 32-36 by at least one plasminogen activator. 38. A host microorganism containing the fusion product obtained in step a) according to the method of any one of claims 1 to 27 or any nucleic acid sequence derived therefrom. 39. The host microorganism according to claim 38, wherein the host microorganism is selected from the following microorganisms: Pichia pastoris, Pichia methanolica, Saccharomyces cerevisiae, Hansenula polymorpha, Aspergillus niger, Aspergillus oryzae or Aspergillus nidulans. 40. Use of functional plasminogen produced by the method according to any one of claims 1 to 27 and/or plasmin obtained therefrom in the preparation of medicaments. 41. The use of functional plasminogen produced by the method according to any one of claims 1 to 27 and/or plasmin obtained therefrom in the preparation of medicines for treating trauma, thrombotic events or preventing thrombotic events application. 42. Use of functional plasminogen produced by the method according to any one of claims 1 to 27 and/or plasmin derived therefrom as an antithrombotic and anticoagulant active agent. 43. The use according to claim 42, especially for the prevention and/or treatment of the following diseases: heart attack, stroke, thrombosis, venous thrombosis, restenosis after heart valve surgery, hypoxia, ischemia, coagulation Acute necrosis, vascular inflammation, acute pulmonary embolism, acute and subacute arterial thrombosis, new or old venous thrombosis, buttock and extremity deep vein thrombosis, early thrombosis in occluded vascular areas, acute ocular central vessel embolism , Conjunctivitis caused by type I plasminogen deficiency, burns, alkali or acid burns and frostbite, disseminated intravascular coagulation during shock, acute extremity arterial embolism, chronic occlusive arterial disease, thrombosis at arteriovenous short circuit; Subsequent treatment of disease: heart attack, bypass surgery, angioplasty, balloon dilatation; thrombolytic therapy in acute heart attack; recanalization of arteriovenous short circuit; reperfusion of blocked coronary artery in acute heart attack . 44. The use according to any one of claims 40 to 43, wherein said functional plasminogen and/or plasmin obtained therefrom, and an anticoagulant are used in combination. 45. The use according to claim 44, characterized in that the anticoagulant is heparin, heparin derivatives or acetylsalicylic acid. 46. Functional plasminogen produced by the method according to any one of claims 1 to 27 and/or plasmin obtained therefrom when added to dressings, ointments or used in combination with wound medicines application. 47. A dressing, a wound bandage or an ointment containing functional plasminogen produced by the method according to any one of claims 1 to 27 and/or plasmin derived therefrom . 48. The dressing, wound bandage or ointment according to claim 47, wherein the pharmaceutical preparation contains 0.01-500 U of plasminogen and/or plasmin derived therefrom, preferably 0.1-250 U, per square centimeter. Particularly preferred is 1-150 U. 49. Use of the dressing, wound bandage or ointment according to claim 47 or 48 in the preparation of a medicament for treating burns, frostbite, alkali or acid burns, injuries and/or wounds. 50. A pharmaceutical composition comprising functional plasminogen produced by the method of any one of claims 1 to 27 and/or plasmin obtained therefrom, and a pharmaceutically acceptable carrier , additives and/or solvents and, if desired, active anticoagulants. 51. The pharmaceutical composition according to claim 50, which is suitable for the following routes of administration: oral, topical or parenteral, intravascular, intraperitoneal, subcutaneous, or intramuscular, said intravascular, preferably intravenous . 52. The pharmaceutical composition as claimed in claim 51, when administered locally, each gram of said pharmaceutical composition contains 0.01-500U plasminogen and/or plasminogen derived therefrom, preferably 0.1-250U, Particularly preferred is 1-150 U. 53. The pharmaceutical composition as claimed in claim 51, when administered orally, each gram of said pharmaceutical composition contains 0.1-100,000 U of plasminogen and/or plasmin derived therefrom, preferably 100-80,000 U U, particularly preferably 1,000-50,000 U. 54. The pharmaceutical composition as claimed in claim 51, when used for injection or transfusion, every 10ml solution contains 0.1-100 million U plasminogen and/or plasmin derived therefrom, preferably 1-10 million U, particularly preferably 3-5 million U. 55. The use of the pharmaceutical composition according to any one of claims 50 to 54 in the preparation of medicaments for the prevention and/or treatment of the following diseases: heart attack, stroke, thrombosis, venous thrombosis, Restenosis after heart valve surgery, hypoxia, ischemia, coagulative necrosis, vascular inflammation, acute pulmonary embolism, acute and subacute arterial thrombosis, new or old venous thrombosis, buttock and extremity deep vein thrombosis , early thrombosis in areas of occluded vessels, acute ocular central vascular embolism, conjunctivitis due to type I plasminogen deficiency, burns, alkali or acid burns and frostbite, disseminated intravascular coagulation in shock, acute extremity arterial embolism, Chronic occlusive arterial disease, thrombosis at arteriovenous short circuit; subsequent treatment of: heart attack, bypass surgery, angioplasty, balloon dilatation; thrombolytic therapy in acute heart attack; Passage; reperfusion of blocked coronary arteries during acute heart attack. 56. A vector comprising the fusion product obtained in step a) of claim 1 or a nucleic acid sequence derived therefrom. 57. A DNA molecule comprising the fusion product obtained in step a) of claim 1 or a nucleic acid sequence derived therefrom. 58. An RNA molecule comprising the fusion product obtained in step a) of claim 1 or a nucleic acid sequence derived therefrom. 59. A screening method for identifying plasminogen activators using the functional plasminogen of claim 36 or 37. 60. The screening method according to claim 59, characterized in that after pre-incubating the protease with the functional plasminogen according to claim 35 or 36, the activity of the obtained plasmin is detected. 61. The screening method according to claim 59 or 60, characterized in that the activity of the obtained plasmin is detected using a synthetic peptide as a substrate. 62. The screening method according to claim 61, characterized in that the activity of the obtained plasmin is detected by using the synthetic peptide N-tosyl-Gly-Pro-Lys-pNA.

Images