MXPA94005661A - Tript inhibitors - Google Patents

Abstract

The present invention relates to novel inhibitors of human tryptase, to its isolation from leeches, to nucleotide sequences encoding the novel inhibitory molecules or fragments thereof, to vectors containing the coding sequence therefor, to host cells transformed with these vectors, to the recombinant production of the inhibitors, to pharmaceutical compositions containing the novel inhibitory molecules, and to their use in the diagnosis and therapeutic

Description

TRIPTASE INHIBITORS INVENTORS: HAMS FRITZ and CH SSTIAN P. SOMMÜ-RHOFF. German citizens, with domicile ßn Am Bucket 33, D-82057 Icking, Germany * and Dßnníngßr Str. 146, D-81927 Münchßn, Germany.

CAUSAHIENT: UCP G1N-PHA1MA AT, a Swiss company, domiciled at Kraftstraße 6, CH - 8044 Zürich, EXTRACT OF THE INVENTION The present invention relates to novel human tryptase inhibitors, to their isolation from leeches, to nucleotide sequences encoding the novel inhibitory molecules or fragments of the myrids, to vectors containing the coding sequence. for them, to host cells transformed with these vectors, to the recoinbinant production of the inhibitors, to pharmaceutical compositions containing the novel inhibitory molecules, and to their use in diagnosis and therapy.

* * * * * The present invention relates to novel inhibitors of human tryptase, to its isolation from leeches, to nucleotide sequences encoding the novel inhibitory molecules or fragments thereof, to vectors containing the coding sequence therefor, to host cells transformed with these vectors, to the recombinant production of the inhibitors, to pharmaceutical compositions containing the novel inhibitory molecules, and to their use in diagnosis and therapy. Tryptase is a tetrameric member of the family of serine proteinases in the form of trypsin. Tryptase is expressed virtually exclusively by sebated cells [Castells Irani, 1987] and is stored in large quantities in its secretory granules, which constitute approximately 23 percent of the total cellular protein [Schwartz Lewis A-usten, 1981]. Following the activation of sebated cells, tryptase is rapidly released into the extracellular space, along with other preformed mediators (eg, histamine, chymase, and proteoglycans) [Schwartz Lewis Seldin, 1981; Caughey Lazarus, 1988]. Elevated levels have been found: ° in the plasma of patients with mastocytosis, after systemic anaphylaxis [Schwartz Metcalfe, 1987; Schwartz Yunginger, 1989], and during the systemic response after aspirin aggression in patients with asthma sensitive to aspirin [Bosso Schwartz, 1991], or in the bronchoalveolar lavage fluid of patients with asthma [Broide Gleich, 1991; Bousquet Chanez, 1991; enzel Fowler, 1988], in the interstitial diseases of the lung [Walls Bennett, 1991], and after the aggression with antigen of allergic patients [Castells, 1988; Butrus, 1990], ° in skin bleb fluid after aggression with cutaneous antigen in patients with atopic and allergic skin disease [Shalit Schwartz, 1990; Atkins Schwartz, 1990], 0 in the nasal lavage fluid after an aggression with local antigen of patients with seasonal allergic rhinitis [Juliusson Holmberg, 1991], ° in the crevicular fluid of patients with gingivitis and periodontitis [Cox Eley, 1989 , J. Period Res; Eley Cox, 1992, J Dent], and 0 in the lesional skin of patients with psoriasis [Harvima Nauk arinen, 1989]. In vitro studies have provided considerable evidence that tryptase is directly involved in the pathogenesis of disorders related to sebaceous cells. For example, tryptase has been suggested as a pathogenic mediator of asthma, since it increases smooth muscle contractility of the airways [Sekizawa, 1989], and inactivates the vasoactive intestinal peptide, thereby destroying its potent bronchodilator action [Tam Caughey, 1990; Tam Franconi, 1990; Franconi, 1989]. In addition, it has been shown that tryptase is a potent mitogen for fibroblasts, suggesting its involvement in pulmonary fibrosis in asthma and interstitial lung diseases.

[Ruoss Hartmann, 1991; Hartmann Ruoss, 1992]. Tryptase has also been implicated in the pathogenesis of arthritis and periodontal diseases, as it activates prostomelysin (= MMP-3), which in turn activates collagenase, thus initiating the destruction of cartilage and periodontal connective tissue, respectively [Gruber Márchese, 1989; Gruber Schwartz, 1990;] Cox Eley, 1989, J Period Res; Eley Cox, 1992, J Dent]. Tryptase can also promote blood coagulation disorders, by inactivating the high molecular weight kininogen procoagulant function [Maier Spragg, 1983] and by dissociating fibrinogen [Schwartz Bradford Litt an, 1985]. Human tryptase is virtually unique among serine proteinases, since it is completely catalytically active in plasma and extracellular space [Schwartz Bradford, 1986; Goldtein Leong, 1992]. Tryptase is not inhibited by naturally occurring antiproteinases that regulate the activity of other serine proteinases in the form of trypsin, such as the mucin proteinase inhibitor (= antileukoprotease or HUSI-I), antithrombin III, alpha1- inhibitor proteinase, alpha2-macroglobulin inhibitor, or ^ -sterase inhibitor [Alter Kra ps, 1990; Smith Hougland, 1984; Schwartz Bradford, 1986; Harvima Schechter, 1988; Cromlish Seidah, 1987], In addition, although tryptase has been known for more than 10 years, derived inhibitors have not yet been described from non-human species, or produced by peptide or recombinant synthesis technologies. Accordingly, tryptase is not affected by hirudin [Alter Kramps, 1990], aprotinin, ovomucoid inhibitor, trypsin inhibitor of soybean and lime bean [Butterfield Weiler, 1990; Cromlish Seidah, 1987; Harvima Schechter, 1988], ecotin [Chung Ives, 1983], and the Kunitz-recombinant domain of Alzheimer's beta-a iloid precursor protein [Sinha Dovey, 1990]. Although tryptase is inhibited by general inhibitors of proteinases in the form of trypsin, such as diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and tosyl-L-lysine chloromethyl ketone [Smitch Hougland, 1984; Harvima Shechter, 1988], these compounds are unsuitable for in vivo applications, and even for most in vitro applications, due to their high toxicity and / or low stability. In addition, the only different inhibitors known to affect tryptase, peptide-arginine aldehydes, leupeptin and antipain [Cromlisch Seidah, 1987], and certain benzamidine derivatives [Stürzebecher Prasa, 1992; Caughey, 1993] are of limited utility, since they are relatively non-specific, and / or inhibit tryptase only with moderate affinities (K values for complexes on the micromolar scale). The problem with the present invention, therefore, is to provide a potent and efficient inhibitor of human proteinase tryptase. As illustrated in more detail below, the present problem can be solved by providing an inhibitory polypeptide obtainable from the medical leech Hirudo medicinalis. Brief description of the figures: Figure 1: isolation of leech-derived tryptase inhibitors, by cation exchange chromatography using SP-Sephadex. The dialyzed leech extract was applied, and the column was washed until absorption of the effluent returned to the baseline. Desorption was achieved with 20 mM NaP, 500 mM NaCl (pH 8.0). Fractions containing the active inhibitor material (labeled with a bar) were pooled. Figure 2: Affinity chromatography of the leech-derived tryptase inhibitors using anhydrotrypsin-Sepharose. The pooled eluate from SP-Sephadex chromatography was applied, the column was extensively washed and eluted with 100 mM KC1 / HC1 (pH 2.1). Fractions containing the active inhibitor material (labeled with a bar) were harvested and neutralized immediately by the addition of 1 M Tris. Figure 3: Cation exchange chromatography of the leech-derived tryptase inhibitors, using a Mono S FPLC column. After dialysis against 20 M NaP (pH 8.0), the pooled eluate was applied from the anhydrotrypsin affinity chromatography, the column was washed, and eluted using a linear gradient of 60 to 240 M NaCl. Fractions containing the active inhibitor material (labeled with a bar) were pooled. Figure 4: SDS-PAGE of the tryptase inhibitors derived from leech isolated under reducing conditions. Track 1 = dialyzed leech extract; lane 2 =: eluate of the SP-Sephadex column; lane 3 = anhydrotrypsin affinity chromatography eluate; lane 4 = eluate of Mono S cation exchange chromatography. Molecular weight markers (lane 5) are from top to bottom: ovalbumin (43 kD), carbonic anhydrase (29 kD), β-lactoglobulin (18.4 kD), lysozyme (14.3 kD), bovine trypsin inhibitor (6200 D), and ß-insulin chain (3400 D). Figure 5: Reversed phase HPLC of isolated leech-derived tryptase inhibitors. The elution time and the absorption of the effluent in 206 nanometers, are given on the abscissa and the ordinate, respectively. The two peaks demonstrate the presence of at least two forms. Figure 6: Tryptic fragmentation of the two species of leech-derived tryptase inhibitors, separated by reverse phase HPLC (see Fiqura 5). Lower trace: HPLC trace of the tryptic digestion of the peak that elutes at 25 minutes in Figure 5; Upper trace: HPLC trace of tryptic digestion of the peak that elutes at 29 minutes. The elution profiles differ only in the peaks representing the C-terminal peptides (marked by the arrows). Figure 7: Mass spectroscopy of the two species of leech-derived tryptase inhibitors, separated by HPLC (see Figure 5). a) the mass spectrum of the HPLC peak eluting after 25 minutes demonstrates the presence of two forms with a mass of 4340 (form A, left peaks), and 4396 (form B, right peaks), respectively. b) the mass spectrum of the HPLC peak eluting at 29 minutes shows a third form (form C), with a mass of 4738. Figure 1: Determination of the sequence of the leech-derived tryptase inhibitors. The bars represent the overlapping fragments used to deduce the amino acid sequence. The solid bars denote the sequence obtained from the type of HPLC eluting at 25 minutes (see Fiqura 5), and the cross bar denotes the additional sequence obtained from the HPLC peak eluting at 29 minutes. N-terminal = sequence obtained from the native inhibitor; Red / T = sequence obtained after reduction and tryptic fragmentation; Ox / T / ChT = sequence obtained after oxidation and tryptic / chymotryptic fragmentation. Figure 9: Inhibition of human tryptase by tryptase inhibitors derived from leech. Tryptase (0.59 nM) was preincubated with increasing concentrations of the leech-derived tryptase inhibitors (0-40 nM) at 37 ° C for 25 minutes, and the reaction was initiated by the addition of the tos-Gly-Pro-Arg substrate. -pNa. The resulting continuous state rates were measured for 3.5 minutes. The given values are the quotient of the speed in the presence of the inhibitor, divided by the speed in the absence of the inhibitor.

Figure 10: Effect of leech-derived tryptase inhibitors on the tryptase-induced dissociation of vasoactive intestinal peptide (VIP). VIP was incubated with tryptase in the presence of increasing concentrations of the leech-derived tryptase inhibitors. Subsequently, the amount of dissociated VIP was quantified by reverse phase HPLC. The given values are the quotient of the speed in the presence of the inhibitor divided by the speed in the absence of the inhibitor. Figure 11: Design, DNA and amino acid sequence of the synthetic rLDTI form C gene. (a) Design of the master gene of form C rLDTI synthetic. The restriction sites entered are shown. (b) Nucleotide and corresponding amino acid sequence of the master form C rLDTI gene. The brackets and numbers indicate the synthetic oligonucleotides used to assemble the gene. (c) Modification of the master gene of form C rLDTI by cartridge mutagenesis. Figure 12: (a) Plasmid map of pRM 5.1.5. (b) pRM expression vector 9.1.4. A synthetic gene for rLDTI-for a C was ligated into a purified yeast secretion vector pVT102U / a, dissociated with Xbal and HindIII. Arrows indicate the direction of transcription; ADH-p, the promoter of the ADH1 gene; mat, the leader gene of coupling factor a; ADH-t, region 31 of the ADH1 gene, including a transcription terminator signal; Ura-3, the Ura gene; amp-R, the ampicillin resistance gene; E. coli ori, yeast ori (2μ-ori), and the intergenic region of phage fl (fl-ori). Figure 13: SDS / PAGE analysis of the fermentation supernatant and form C rLDTI purified track 1 - markers of low molecular mass; lane 2 = fermentation supernatant of yeast strain H005 after 96 hours of culture (80 μl); lane 3 = rLDTI form C purified (2 μg). Figure 14: HPLC analysis of purified rLDTI-for a C. Reverse phase HPLC on a RP 18 column, which was performed with 7.4 nanomoles (35 micrograms) of purified inhibitor. A linear gradient of 0 to 60 percent (by volume) of acetonitrile was formed, from 0.1 percent (by volume) of trifluoroacetic acid to acetonitrile, and 0.1 percent (by volume) of trifluoroacetic acid was used. The flow rate was adjusted to 1.0 milliliters / minute, and the absorbance in the effluent was monitored at 206 nanometers. Figure 15: Plasmid map of the expression vector pRM 3.1.10. Figure 16: Plasmid map of the expression vector pRM 4.1.4. Figure 17: Plasmid map of the expression vector pRM 11.1.4. In accordance with a first modality, the present invention relates to purified inhibitory molecules of human tryptase. The novel inhibitors are polypeptides that can be obtained from extracts of leeches, such as, for example, the medical leech Hirudo medicinalis. The present invention also relates to the functional equivalents of the inhibitory molecules that show tryptase inhibitory activity, and to the pharmaceutically acceptable salts of the inhibitors. The inhibitor molecules of the present invention are characterized by their ability to inhibit human tryptase with a K value on the scale of about 0.1 to 10 nM, leaving the proteases involved in the human blood coagulation cascade, substantially unaffected. Preferably, tryptase inhibitors are provided which are characterized by the following amino acid sequence (position 1: N-terrainal amino acid Lys): Lys-Lys-Val-Cys-Ala-Cys-Pro-Lys-Ile-Leu 10 Lys- Pro-Val-Cys-Gly-Ser-Asp-Gly-Arg-Thr 20 Tyr-Ala-Asn-Ser-Cys-Ile-Ala-Arg-Cys-Asn 30 Where X = H (SEQUENCE IDENTIFICATION NUMBER: 1 ), Gly (SEQUENCE IDENTIFICATION NUMBER: 2) or Gly-Ile-Leu-Asn (SEQUENCE IDENTIFICATION NUMBER: 3). The present invention also encompasses genetic variants, alleles or functional equivalents of the aforementioned sequence, from which one or more of the amino acids are substituted (conservative or non-conservative), or deleted, or to which one or more of the amino acids are added. amino acids without substantially affecting the inhibitory activity of tryptase. Conservative substitutions encompass, for example, substitutions within the following groups of amino acids (one letter code): G, A; VILE; FROM; N, Q; K, R; and S, Y, N. Preferably, the addition or deletion of amino acids is carried out at the N- and / or C-terminal end of the aforementioned sequence. Functional equivalents with an altered and / or improved specificity, and / or an inhibitory efficiency, can be readily prepared by a person of ordinary experience, by applying the usual methods of peptide synthesis, or by applying well-known methods in the field of molecular biology, such as, for example, site-directed mutagenesis, or non-targeted mutagenesis (e.g., using a phage visual display system). The functional equivalents of the inhibitor of the present invention are, for example, those comprising the amino acid sequence: R1-Cys-Pro-Lys-Ile-Leu Lys-Pro-Val-Z-Gly-Ser-Asp-Gly-Arg -Thr Tyr-Ala-Asn-Ser-Cys-Ile-Ala-R2 wherein: the N-terminal residue R1 represents Ala- or Cys-Ala-; the N-terminal residue R2 represents -Arg or -Arg-Cys; Z defines any, preferably any amino acid that occurs naturally. Moreover, based on the description of the present invention, a person of ordinary experience will be able to prepare fragments of the natural forms of the inhibitor, still showing the desired tryptase inhibitory activity. The naturally occurring forms of the claimed inhibitory molecules can be isolated from leech, preferably the medical leech Hirudo medicinalis, or they can be prepared by peptide synthesis or recombinant DNA technology. In accordance with a further aspect of the present invention, a synthetic gene encoding form C (SEQUENCE IDENTIFICATION NUMBER: 3) of the leech-derived tryptase inhibitor (LDTI-C) was designed, cloned, and expressed. , in Escherichia coli and Saccharomyces cerevisiae. The coding fragment was assembled by means of 6 oligonucleotides, contains linker sequences, stop codons, and recognition sites selected for other modifications, for example, by cartridge mutagenesis. The strong expression of the recombinant C-form inhibitor (rLDTI-C) was found using the secretion vector of Saccharomyces cerevisiae pVT102U / alpha, and strain S-78. The secreted material was isolated by centrifugation and cross-flow filtration, and further purified by cation exchange chromatography, it is inhibitoryly active, and approximately 85 percent pure. The amino acid sequencing showed that the rLDTI-C is processed in a predominantly correct manner at the junction between the leader peptide of the alpha coupling factor and the first amino acids of LDTI-C; only minor amounts of truncated forms were detected. The UV-CD far spectrum of the recombinant molecule is typical for a bent protein containing secondary structural elements. The molecular mass of the material purified by HPLC is 4738 + 4 Da, determined by electrospray ionization mass spectrometry. RLDTI-C exhibits dissociation constants in equilibrium with bovine trypsin and human tryptase, which are almost identical to the natural ones. The expected expression products encoded within the expression vector were also identified in vitro, using an S30 transcription transfer system. The proteins presented in the present invention are the first compounds that are efficient tryptase inhibitors. Accordingly, the leech-derived triptase inhibitors reduce the catalytic activity of the tryptase, the K value of the enzyme inhibitor complex being on the nanomolar scale. Moreover, the inhibitors affect not only the tryptase-induced dissociation of the nitroanilide substrate of the peptide used co or a tool to determine the activity of the proteinase in vitro. They also affect the dissociation of vasoactive intestinal peptide (VIP) and kininogen, representative of peptides and proteins thought to be biologically relevant substrates of tryptase. In addition, the inhibitors efficiently decrease the tryptase-induced growth of human keratinocytes - an example of the direct cellular effects of tryptase - without causing cytotoxic effects or other apparent side effects. In addition to having a high affinity for tryptase, tryptase inhibitors derived from leeches are highly effective. Therefore, with the exception of the pancreatic proteinases trypsin and chemotrypsin, other serine proteinases do not inhibit or are only marginally inhibited, with the values Kj ^ for the enzyme-inhibitor complexes being at least 200 times higher than for the tryptase complex. . Its specificity is illustrated by the effect of high blood pressure on ex vivo blood coagulation, verifying that the proteinases involved in the coagulation cascade are not affected. Therefore, the leech-derived tryptase inhibitors of the present invention will allow for the first time the inhibition of tryptase with a high affinity and specificity. Accordingly, the inhibitors provide the prospect of effectively blocking the pathophysiological events involving the dissociation of proteins and peptides and / or the activation of cells by tryptase. Accordingly, it is an object of the present invention to apply the inhibitors as probes in diagnosis, as well as drugs in the therapy of diseases related to tryptase and sebaceous cells. In accordance with a further preferred embodiment of the present invention, nucleotide sequences, such as, for example, DNA and RNA sequences, which encode a polypeptide with tryptase inhibitory activity or fragments thereof, are provided. Preferably, polynucleotide molecules are provided which comprise the following general nucleotide sequence (SEQUENCE IDENTIFICATION NUMBER: 4): 5 '1 AARAARGTNTGYGCNTGYCCNAARATHYTNAARCCNGTNTGYGGNWSNGA 51 YGGNMGNACNTAYGCNAAYWSNTGYATHGCNMGNTGYAAYGGNGTNWSNA 101 THAARWSNGARGGN SNTGYCCNACNX 3 'where R denotes A or G; M denotes A or C; W denotes A or T; S denotes C or G; And denotes C or T; H denotes A, C, or T; N denotes any nucleotide; and X denotes -OH (SEQUENCE IDENTIFICATION NUMBER: 4), GGN (SEQUENCE IDENTIFICATION NUMBER: 5), or GGN ATH YTN AAY (SEQUENCE IDENTIFICATION NUMBER: 6). The present invention also relates to the complementary cord thereof; and DNA sequences that hybridize, preferably under stringent conditions, in the aforementioned DNA sequence. Preferably, the polynucleotides of the present invention comprise a nucleotide sequence that substantially corresponds to nucleotide residues 1 to 149, or more preferably 7 to 144 of SEQUENCE IDENTIFICATION NUMBER: 7; or fragments thereof comprising at least 15 to 21 consecutive nucleotides of the SEQUENCE IDENTIFICATION NUMBER: 7. Within the scope of the present invention, there are also complementary polynucleotides comprising a nucleotide sequence substantially corresponding to the residues of nucleotide 1 to 149, or preferably 10 to 147 of the SEQUENCE IDENTIFICATION NUMBER: 8; or fragments thereof comprising at least 15 to 21 consecutive nucleotides of the SEQUENCE IDENTIFICATION NUMBER: 8. Preferably, these fragments are specific or functional tryptase inhibitor derivatives of these nucleotide sequences. According to a further embodiment, the present invention relates to an oligonucleotide that hybridizes, preferably under stringent conditions, to a nucleotide sequence encoding a polypeptide with a tryptase inhibitory activity. Preferably, this oligonucleotide comprises a nucleotide sequence that is substantially complementary to the nucleotide sequence of residue 22 to residue 87 of the SEQUENCE IDENTIFICATION NUMBER: 7. Another embodiment of the present invention relates to polynucleotides that encode a polypeptide having activity. Tryptase inhibitor, whose polynucleotides can be obtained by hybridization, preferably under stringent conditions, with an oligonucleotide as specified above; as well as to polypeptides encoded by these polynucleotides. The appropriate strict conditions can easily be determined by an expert in this field. Also within the scope of the present invention is the polynucleotide sequence of the SEQUENCE IDENTIFICATION NUMBER: 9, and its functional equivalents. The present invention also encompasses vector molecules for the transformation of eukaryotic or prokaryotic hosts, comprising a DNA molecule as defined above. For example, the vector may be a virus or a plasmid containing the DNA sequence encoding the inhibitor in a functional relationship with suitable transcriptional and translational regulatory sequences well known in the art. The coding sequence can also be linked with suitable autonomous replication sequences (ARSS). Suitable host cells can be transformed with a vector containing the coding sequence of the tryptase inhibitor, and the inhibitor produced by the host cells can be expressed and isolated in a suitable manner from the cell culture. A further embodiment of the present invention is a polypeptide expression cartridge comprising a promoter operably linked to a DNA sequence encoding the polypeptide, and to a DNA sequence containing transcription termination signals. In the host capable of secreting the expressed polypeptides, the expression cartridge preferably comprises a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the appropriate reading frame with a second DNA sequence encoding the polypeptide of the invention, and a DNA sequence containing transcription termination signals. In a preferred embodiment, the promoter, the signal sequence, and the terminator are recognized by the expression system of the yeast. The promoters suitable for expression in a certain host are well known. Examples are the promoter of the TRP1 gene, the ADHI or ADHII gene, the acid phosphatase gene (PH05), the CUP1 gene, the isocitochrome c gene, or a promoter of genes encoding glycolytic enzymes, such as TDH3, dehydrogenase of glyceraldehyde-3-phosphate (GAPDH), a shortened version of GAPDH (GAPFL), 3-phosphoglycerate (PFK) kinase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, Pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, invertase and glucokinase genes, or a promoter of the yeast coupling pheromone genes encoding factor a or a, can be used. Preferred vectors of the present invention contain, for example, promoters with a transcription control that can be activated or deactivated by varying the growth conditions, for example, the promoter of the PH05 or CUP1 gene. For example, the PH05 promoter can be repressed or derepressed at will, exclusively by increasing or decreasing the concentration of inorganic phosphate in the medium, and the CUP1 promoter can be activated by the addition of Cu2 + ions to the medium, for example, in the form of a copper salt. GAPDH and the yeast CUP1 promoter are especially preferred. The DNA sequence encoding a signal peptide ("signal sequence"), eg, a yeast signal peptide, is preferably derived from a gene, eg, a yeast gene, which codes for a polypeptide that is ordinarily secreted. The yeast signal sequences are, for example, the signal sequences and prepro of the invertase yeast (SUC2), factor, pheromone peptidase (KEX1), "annihilating toxin" and repressible acid phosphatase (PH05), and the sequence of Glucoamylase signal from Aspergilluss awamori. Additional sequences, such as pro or spacer sequences, which can carry specific processing signals in the constructs, can also be included to facilitate accurate processing of the precursor molecules. For example, the processing signals contain a Lys-Arg residue, which is recognized by a yeast endopeptidase located in the Golgi membranes. Preferred signal sequences according to the present invention are those of the yeast PH05 gene, the factor, and the yeast invertase gene (SUC2). A DNA sequence containing transcription termination signals, eg, yeast transcription termination signals, is preferably the sequence of the 3 'flank of a gene, eg, a yeast gene, containing signals appropriate for transcription termination and polyadenylation. The preferred flanking sequence is that of the PHOS yeast and the factor a gene. The DNA encoding the polypeptide according to the present invention can be isolated from a gene bank of the natural host (the medical leech Hirudo medicinalis), by methods known in the art, or it can be synthesized by PCR used, by example, the use of the host's preferred codon. The promoter, the DNA sequence encoding the signal peptide, the DNA sequence encoding the polypeptide, and the DNA sequence containing the transcription termination signals, are operably linked to one another, ie, they juxtapose in such a way that their normal functions are maintained. The arrangement is such that the promoter makes an appropriate expression of the signal sequence complex-polypeptide gene, and the transcription termination signals effect an appropriate termination of the transcription and polyadenylation. The signal sequence is linked in the appropriate reading frame with the polypeptide gene, such that the last codon of the signal sequence is directly linked to the first codon of the gene for the polypeptide. The yeast promoter preferably binds to the signal sequence between the main mRNA start, and the ATG naturally linked to the promoter gene. The signal sequence has its own ATG for the initiation of the transfer. The binding of these sequences, for example, can be effected by means of synthetic oligodeoxynucleotide linkers carrying the recognition sequence of an endonuclease. Examples for the related expression cartridges are described, for example, in EP-A-341215. Preferred expression cartridges include the CUP1 or GAPDH promoter, the factor, or the yeast invertase leader sequence, the tryptase inhibitor gene, and the factor a terminator. The especially preferred expression cartridge includes a recombinant DNA molecule, as described in Example 9, or a functional fragment or derivative thereof. A further embodiment of the present invention relates to a recombinant plasmid comprising a polypeptide expression cartridge as described above. Apart from the polypeptide expression cartridge, the expression plasmids according to the present invention can include a DNA segment that originates from two-micron DNA that contains the replication origin, or if a DNA-free yeast strain is used. of two microns, DNA of two microns in total. The last type of plasmid is preferred. For example, the plasmids according to the present invention can contain the DNA of two full micras in an uninterrupted form, ie, the DNA of two micras is dissociated once with a restriction endonuclease, the linearized DNA is linked with the others vector components before recirculation. The restriction site is selected such that normal function of the REP1, REP2, and FLLP genes is maintained, and of the ORÍ, STB, IR1, and IR2 sites of two-micron DNA, as well as the small "target" sites. FLP recognition "(FRT), located near the center of each inverted repeat (IR) where the FLP recombinase acts. Optionally, the restriction site is selected such that the D gene of the two-micron DNA remains intact as well. Suitable restriction sites are, for example, the unique PstI site located within the D gene, and the unique HpaJ and SnaBI sites located outside of all genes and sites. However, in the same way it is possible to insert the expression cartridge and other components (cf below) into different (such as two) restriction sites, especially those mentioned above, into the DNA of two micras. Preferably, the expression plasmids according to the present invention include one or more, especially one or two, selective genetic markers, for example, a marker for yeast and a marker and (except for the hybrid vectors in the form of two symmetric microns). ) a replication origin for a bacterial host, especially Escherichia coli. With respect to the selective genetic markers, any marker gene that facilitates the selection for the transformants can be used, due to the phenotypic expression of the marker gene. Suitable labels are, for example, those that express resistance to the antibiotic, or in the case of the auxotropic yeast mutants, the genes that complement the host lesions. Corresponding genes confer, for example, resistance to the antibiotics G418, hygromycin, or bleomycin, or provide a prototrophy in the auxotrophic yeast mutant, for example, the URA3, LEU2, LYS2, HIS3 or TRP1 gene. Since the amplification of the expression plasmids is conveniently carried out in a prokaryote, such as E. coli, a prokaryote is conveniently included in the replication source, for example, E. coli, or a genetic marker and a prokaryote, for example, E. coli These can be obtained from the corresponding prokaryotic plasmids, for example, E. coli plasmids, such as pBR322 plasmid or a pUC plasmid, for example, pUC18 or pUC19, both containing a prokaryotic replication origin, for example, JE -, coli, and a genetic marker that confers resistance to antibiotics, such as ampicillin. A suitable vector for transforming yeast cells is the plasmid pRM 9.1.4, as deposited with the lbSM, and having a DSM accession number 9271. Other preferred vector molecules are: pRMll.1.4, as deposited with the DSM , and that they have the access number DSM 9272; pRM5.1.5 as it is deposited with the DSM, and that have the access number DSM 9270; pRM4.1.4 as it is deposited with the DSM, and that have the access number DSM 9269; pRM3.1.10 co or is deposited with the DSM, and that have the access number DSM 9268; The vector can also be selected from pHE175, pHE175R, pHE177 and pHE177R, as described in the experimental part below. According to a further embodiment of the present invention, there is provided a method for preparing a tryptase inhibitor, which includes the steps of: a) obtaining an extract from a leech, preferably from the medicinal leech Hirudo medicinalis, and b) purifying the extract by dialysis and column chromatography. Preferably, the crude extract is dialyzed against a regulator of low ionic strength. Subsequently, the dialyzed extract is purified by cation exchange chromatography, biospecific chromatography, such as, for example, anhydrotrypsin-sepharose affinity chromatography and another step of cation exchange chromatography. Other experimental details are illustrated in the experimental part that follows. The modifications of the claimed process can be easily designed by a person of ordinary experience, which are still encompassed by the scope of the present invention. The present invention also relates to methods for preparing a recombinant tryptase inhibitor, and recombinant tryptase inhibitors that can be obtained by these methods. In accordance with a preferred embodiment, this method includes: a) transforming a prokaryotic or eukaryotic host with a vector as defined above; b) inducing the expression of the coding sequence of the tryptase inhibitor; c) recovering the expression product; and optionally d) removing from the obtained product peptide fragments not required for the activity of the tryptase inhibitor and / or optionally renaturing the product. The coding sequence can also be expressed by the application of a suitable transcription-tracing system, such as, for example, the S30 transcription transfer system. In accordance with another embodiment of the present invention, prokaryotic and eukaryotic hosts transformed with a vector encoding a tryptase inhibitor, and variants and mutants thereof are provided. Suitable hosts are of prokaryotic or eukaryotic origin. Examples are bacterial, fungal, plant, or insect cells. Preferred hosts are bacterial and fungal cells, such as E. coli, or fungi, Saccharomyces cerevisiae, Aspergillus niger, Aspergillus nidulans or Neurospora crassa. Preferred yeast strains are those mentioned above, for example, S. cerevisiae, which have been cured of the endogenous two-micron plasmid ("cir0 strains"), and especially strains that are simply or multiply deficient in yeast proteases; and / or, in the case where the CUP1 promoter is used, yeast strains containing from 1 to 3 additional copies of the chromosomal CUP1 gene. A wide variety of proteinases, such as those mentioned, have been characterized in the yeast Saccheiromyces cerevisiae, [Aschstetter et al. (1985)]. Mutants lacking the activity of most of these proteases have been isolated and studied biochemically. The consequences of the absence of certain proteases were elucidated, and some properties proved useful for the production of heterogeneous proteins. The proteases that are lacking in the yeast strains according to the present invention, do not perform indispensable functions in the cellular metabolism; therefore, mutations that completely destroy the activity of these proteins are not lethal. For example, the yeast strain lacks one or more proteases selected from the group of carboxypeptidases ysca-, yscB, yscA, yscY and yscS. Methods for the production of these yeast strains are described, for example, in EP-A-40170 and EP-A-341215. The transformation of the host with the hybrid plasmids according to the present invention, it can be done in accordance with the known methods C I lu technique. A preferred embodiment refers to a eukaryotic host derived from S. cerevisiae S-78, as deposited with the DSM, and having the accession number DSM 9273, and variants and mutants thereof, capable of producing a tryptase inhibiting molecule. In accordance with another preferred embodiment of the present invention, pharmaceutical compositions are provided, including a tryptase inhibitory amount of a polypeptide as defined above, prepared from leech extracts, or obtained, for example, by the expression of a encoding gene for recombinant tryptase inhibitor, optionally in combination with a pharmaceutically acceptable carrier or diluent. These compositions can be used in particular in the case of the indications mentioned herein, if they are administered, for example, parenterally (such as intravenously, intracutaneously, intramuscularly, or subcutaneously), orally, by inhalation, or locally. The dosage depends essentially on the specific method of administration and the purpose of the treatment or prophylaxis.

The size of the individual doses and the administration schedule can best be determined based on an individual assessment of the relevant case. The methods required to determine the relevant factors are familiar to the expert. Normally, in the case of injection, the therapeutically active amount of the compounds according to the present invention is in the dosing scale from about 0.005 to about 1 milligram / kilogram of body weight. The scale of about 0.01 to about 0.05 milligrams / kilogram of body weight is preferred. Administration is by intravenous, intramuscular, or subcutaneous injection. Accordingly, depending on the method of application, pharmaceutical preparations for parenteral administration contain about 0.05 to about 10 milligrams of the compound according to the present invention per individual dose. In addition to the active ingredient, these pharmaceutical compositions usually also contain a regulator, for example, a phosphate buffer, intended to maintain the pH value between about 3.5 and 7, and in addition, sodium chloride, mannitol, or sorbitol, with the object to adjust the isotonicity. They may be in a freeze-dried or dissolved form, wherein the solutions may conveniently contain an antibacterial preservative, for example, 0.2 to 0.3 percent methyl ester or 4-hydroxybenzoic acid ethyl ester. A preparation for local application may be in the form of an aqueous solution, lotion or jelly, an oily solution or suspension, or a fatty ointment, or particularly in emulsion. A preparation is obtained in the form of an aqueous solution, for example, by dissolving the substance according to the present invention, or a therapeutically useful salt thereof, in an aqueous buffer solution of a pH of from 4 to 6.5, and if you want, you add one or more additional substances to it. The concentration of the active ingredient is from about 0.08 to about 1.5 milligrams, preferably from 0.25 to 1.0 milligrams, in about 10 milliliters of a solution, or 10 grams of a jelly. An oily form of application for local administration is obtained, for example, by suspending the substance according to the present invention, or a therapeutically useful salt thereof in an oil, optionally with the addition of swelling agents, such as aluminum stearate. , and / or surface activity agents (surfactants), whose HLB (hydrophilic-lipophilic balance) value is less than 10, such as fatty acid monoesters of polyhydric alcohols, for example, glycerol monostearate, sorbitan monolaurate, monostearate sorbitan, or sorbitan monooleate. A fatty ointment is obtained, for example, by suspending the substance according to the present invention, or the salts, in an extensible oily base, optionally with the addition of a surfactant having a HLB value of less than 10. obtains an emulsion ointment by triturating an aqueous solution of the substance according to the present invention, or the salts, in an expandable soft greasy base, with the addition of a surfactant, whose HLB value is less than 10. All these Local application forms may also contain a preservative agent. The concentration of the active ingredient is from about 0.08 to about 1.5 milligrams, preferably from 0.25 to 1.9 milligrams in about 10 grams of the matrix. The present invention also relates to the bioanalytical use of the compounds according to the present invention and the salts thereof, for the analytical determination of tryptase, and the preparations which serve for this purpose, which contain the substances according to the present invention. invention, for example, solid mixtures and all previous solutions, in particular, aqueous solutions. In addition to the specific amount or concentration of the substances according to the present invention (also in the form of a salt), these may also contain inert adjuvants, for example, those mentioned above with reference to the preparations for injection, having , for example, a stabilizing and / or conservative function. According to a further embodiment, the present invention relates to the use of a tryptase inhibitor as defined above, in the diagnosis of disorders related to functional tryptase and sebaceous cells. Especially preferred is the use for the preparation of pharmaceutical compositions for the treatment of asthma, interstitial lung disease, arthritis, periodontal disease, allergic disorders, disorders of blood coagulation, skin disorders, and psoriasis.

EXPERIMENTAL PART 1. MATERIALS a) Leech extracts: the extracts of the leech Hirudo medicinalis, were a gift from Plantorgan, Germany. The leech extracts can also be prepared based on the description of EP-A-0 207 956, and the references cited therein. b) Enzymes and substrates: the proteases were obtained as follows: bovine trypsin, porcine pancreatic kallikrein, and porcine pancreatic elastase (Sigma, Deisenhofen, Germany); human Xa factor (Boeringer; Mannheim, Germany); human neutrophil elastase, human thrombin, human urokinase, and bovine chymotrypsin (Medor; Hersching, Germany); human plasmin, and human plasma kallikrein (Kabi; Essen, Germany); human cathepsin G (Calbiochem, Bad Soden, Germany). Tryptase was purified from human lung tissue to apparent homogeneity using a modification of the described methods [Smith Hougland, 1984; Schwartz Lewis Austen, 1981; Harvima Schechter, 1988]. The following substrates were purchased: Bz-Ile-Glu-Gly-Arg-pNA (Novabiochem; Bad Soden, Germany); Suc-Ala-Ala-Ala-pNA (Bachem; Heidelberg, Germany); D-Pro-Phe-Arg-pNA, and D-Val-Leu-Arg-pNA (Kabi, Essen, Germany); Suc-Val-Pro-Phe-pNA, and Pyr-Gly-Arg-pNA (Medor; Herrsching, Germany); MeO-Suc-Ala-Ala-Pro-Val-pNA (Sigma, Munich, Germany). Tos-Gly-Pro-Arg-pNA was obtained from Boehringer, Mannheim, Medor, and Sigma. (Cough = Tosyl; Suc = succinyl; "pNA" = p-nitroanilide). Vasoactive intestinal peptide (VIP) was purchased from Calbiochem (Bad Soden, Germany), and bovine lung heparin from Sigma. The Bdellin B was a gift from E. Fink (Kllinische Chemie und Klinische Biochemie, Chirurgische Klinik, LMU, Munich, Germany). c) Column materials: SP-Sephadex®, Sepharose® 4B activated with cyanogen bromide, and Mono S® HR 5/5 were obtained from Pharmacia (Freiburg, Germany). Anhydrothrypsin was prepared from trypsin, purified by affinity by a modification of the methods described by Ako [Ako Foster Ryan, 1972] and immobilized on Sepharose 4B activated with cyanogen bromide in accordance with the Pharmacia guidelines. d) Cell culture: The medium, fetal calf serum, and antibiotics were obtained from Biochrom (Berlin, Germany). The human keratinocyte cell line HaCaT [Boukamp Petrussevska, 1988] was obtained from N. Fusenig, German Cancer Research Center (DKFZ, Heidelberg, Germany). The [Aethyl-3H] shy was purchased from Amersham Buchler (Braunschweig, Germany). 2. METHODS 2.1 Purification of the leech-derived tryptase inhibitor a) SP-Sephadex® chromatography: The leech extract (approximately 3.5 grams) was dissolved in deionized water (77 milliliters), and dialysed against 20 nM NaP (pH 8.0) overnight at 4 ° C. The dialyzed material was applied on a SP-Sephadex® column (1.6 x 20 centimeters) balanced with the same regulator. The column was washed at a flow rate of one milliliter / minute, until the optical density (280 nanometers) of the effluent reached the baseline, and was eluted with 20 mM NaP, 500 mM NaCl (pH 8.0) . The fractions containing the active inhibitor material were collected and pooled. b) Affinity chromatography on anhydrotripsin-Sepharose R: The material pooled from cation exchange chromatography (approximately 20 milliliters), was applied on an anhydrotrypsin-Sepharose column (1.6 s 3.6 centimeters) equilibrated with 20 mM NaP ( pH of 8.0). About 90 percent of the active inhibitor material was bound; the rest of the flow was collected for rechromatography. After extensive washing of the column (approximately 10 column volumes), elution was initiated by the addition of 100 mM KC1 / HC1 (pH 2.1), at a flow rate of 0.3 milliliters / minute. The fractions were harvested and immediately neutralized by the addition of 1 M Tris. The pooled eluate was dialyzed against 20 mM NaP (pH 8.0) overnight at 4 ° C. c) Chromatography on Mono SR: Eluate dialyzed from affinity chromatography, was bound on a Mono SR cation exchange column (0.5 x 5 centimeters) equilibrated with 20 mM NaP (pH 8.0). The column was washed with the same regulator (approximately 20 milliliters), and eluted using a gradient of 60 to 240 mM NaCl in 50 column volumes at a flow rate of 1 milliliter / minute. Fractions containing the active inhibitor material (approximately 5 milliliters) were pooled, aliquots formed, and stored at -20 ° C. 2. 2. Standard Analytical Methods a) Protein assay: Protein concentrations were determined using the bicinic acid method [Smith Krohn, 1985], with bovine serum albumin as standard. b) Electrophoresis: The electrophoretic analysis of the reduced and denatured protein was carried out using 10 to 20 percent of SDS-polyacrylamide gradient gels, as described by Laemmii [Laemmii, 1970]. Proteins were detected after staining with silver [Huekeshoven, 1985]. c) HPLC: Samples (about 1 nanomole) were loaded onto a Lichrospher RP 8 reverse phase column (120 x 4 millimeters, Merck), and eluted using a linear gradient from 0 percent to 30 percent acetonitrile in 0.1 percent of TFA, at a flow rate of 1 milliliter / minute. d) Sequence analysis: Reduction and S-β-pyridylethylation: S-β-pyridylethylation was performed essentially as described by Friedman et al [Friedman Krull, 1970]. The inhibitor (1-2 nanomoles) was dissolved in 100 microliters of regulator (6 M guanidinium HCl, 0.25 M Tris-HCl, 1 mM EDTA, 5 percent (volume / volume) of β-mercaptoethanol; 8.5), and incubated overnight at room temperature. After addition of 5 microliters of 4-vinylpyridine, and incubation for 90 minutes, the reaction was stopped by acidification with formic acid. The S-pyridinethylated inhibitor was desalted by reverse phase chromatography on an Aquapore RP 300 column (2.1 x 30 millimeters, Applied Biosystems, Pfungstadt, Germany). Oxidation of the inhibitor: A mixture of formic acid (45 microliters) and acid peroxide (30 percent, 5 microliters) was preincubated for one hour at room temperature. Subsequently, the inhibitor (1-2 nanomoles) was dissolved in this mixture. After incubation for 1 hour at 4 ° C, the reaction was stopped by dilution with one milliliter of deionized water and lyophilization. Fractionation: The inhibitor (1 nanomole) was incubated with trypsin and / or chymotrypsin (both sequencing grade, Boeringer, Mannheim) in 100 microliters of 1 M ammonium acid carbonate buffer (pH 8.0) for 14 hours at 37 ° C. An enzyme / inhibitor ratio of 1:40 was used. The reaction was terminated by acidification with formic acid, and the fragments were separated by HPLC. Amino acid sequence analysis: Automated amino acid sequencing was performed using a 473A gas phase sequencer (Applied, Biosystems, Weiterstadt, Germany). e) Sequence comparison: The MIPSX database (Martinsrieder Institut für Proteinsequenzen am Max-Planck-Institut für Biochemie, Martinsried, Germany) was investigated using the fast protein search algorithm Lipman & Pearson, FASTP [Lipman Pearson, 1985]. The alignments were optimized using CLUSTAL [Higgins Sharp, 1988]. f) Amino acid analysis: The samples of the oxidized inhibitor were hydrolysed under vacuum in 5.7 M hydrochloric acid at 110 ° C for 20 hours, and analyzed on a high performance Biotronik LC 5000 analyzer system (Puchheim, Germany). g) Determination of the molecular mass: The molecular mass of the purified HPLC inhibitor (50 μM) was determined using an API III row quadrupole instrument (Sciex, Thornhill, Ontario, Canada). The instrument was calibrated with polypropylene glycol ammonium adduct ions. h) Inhibitory activity: During the purification procedure, the inhibitor was followed by measurements of its effect on the idolitic activity of tryptase. Accordingly, the samples were incubated with tryptase (0.59 nM) in 50 mM Tris / HCl (pH of 7.6), 150 mM NaCl, 50 micrograms / milliliters of bovine lung heparin, and 0.1 percent (weight / volume) of bovine serum albumin, for 25 minutes at 37 ° C. The assay was initiated by the addition of the tos-Gly-Pro-Arg-pNa substrate, to a final concentration of 0.1 mM. Released nitroaniline was monitored spectrometrically at 405 nanometers for 3.5 minutes using a UVIKON 930 photometer (Kontron; Eching, Germany). An inhibition unit (IU) was defined as the amount of inhibitor that reduces substrate hydrolysis by 30 percent. i) Titration of the inhibitor: The concentration of the tryptase inhibitor derived from active leech inhibitor was determined by titration with trypsin. Accordingly, bovine pancreatic trypsin was standardized by active site titration using p-Nitrophenyl p'-guanidinobenzoate [Chase Shaw, 1970). The concentration of the active inhibitor was calculated assuming a 1: 1 interaction between the inhibitor and trypsin. k) Determination of equilibrium constants: To determine the specificity of the inhibitor, its effect on the amidolytic activity of different serine proteinases was determined (see Table 5). Accordingly, the proteinases were incubated with the inhibitor (0.2 μM) for 15 and 30 minutes, under the conditions indicated in Table 5. The residual enzymatic activity was measured after the addition of a suitable substrate. The equilibrium dissociation constants (KL) for the inhibitor complexes with individual proteases were determined essentially as described by Bieth [Bieth, 1980]. Briefly, increasing concentrations of the inhibitor were incubated with a constant concentration of the enzyme; the time necessary to reach equilibrium of the enzyme-inhibitor complex for each protease was determined in the preliminary experiments. Then the substrate was added, and the residual enzymatic activity was measured. Values were calculated by adjusting the continuous state velocities to the equation for tight binding inhibitors [Morrison, 1969] using non-linear regression analysis. 1) Coagulation assay: Prothrombin time according to Quick and partial thromboplastin time were measured, using an Amelung KC 10 coagulometer (Lemgo, Germany), and the reagent series from Behringwerke AG (Marburg, Germany), from compliance with the manufacturers' guidelines. ) Dissociation of vasoactive intestinal peptide (VIP): Tryptase (4.8 nM) was preincubated with different concentrations of the leech-derived tryptase inhibitor in 100 mM Tris (pH 7.4), 140 mM NaCl, 50 micrograms / milliliter heparin at 37 ° C for 25 minutes. Then the vasoactive intestinal peptide (VIP, 24 μM, final concentration) was added. After incubation for 1 to 10 additional minutes, the reaction was stopped by acidification with acetic acid. The remaining VIP and the fragments generated were quantified using HPLC. n) Growth of human keratinocytes; For growth studies, the human keratinocyte cell line HaCaT, a spontaneously transformed cell line that maintains the characteristics of differentiated keratinocytes, was used [Boukamp Petrussevska, 1988]. The HaCaT cells were coated on 24-well tissue culture plates. (Falcon; Becton Dickinson, Heidelberg, Germany), at a density of 104 cells / square centimeter, in a medium containing 90 percent Dulbecco's modified Eagle's medium, 10 percent fetal calf serum, and 50 micrograms / milliliter of gentamicin. The cells were incubated at 37 ° C in 5 percent C02. After 24 hours, the cells were washed twice with serum-free Dulbecco modified Eagles medium, and fresh serum-free medium containing 7.8 micrograms / milliliter of heparin alone, or medium containing the agonists and / or the inhibitor was added. After 48 hours, the cells were washed twice again, and fresh serum-free medium containing 1 μCi / ml of 3H-thymidine was added. After an additional two hours, the cells were washed three times with ice-cold Dulbecco PBS, and the incorporated 3H-thymidine was precipitated by 10 percent trichloroacetic acid. After the solubilization of the precipitate in 0.1 N NaOH, 1 percent SDS, the incorporated radioactivity was determined by liquid scintillation count (beta counter model LS 1800, Beckman Instruments, Munich, Germany). For growth studies, other non-keratinocyte cell lines may also be applied.

Example 1: Isolation of the leech-derived tryptase inhibitor 3.5 grams of lyophilized leech extract were dissolved in water, dialyzed against 20 mM NaP (pH 8.0), and applied on a SP-Sephadex® cation exchange column ( see the Method Section). The volume of the protein (approximately 98 percent), and the trypsin inhibitory activity, were found in the flow through, while the leech-derived tryptase inhibitor was bound to the column. After elution of the column with 500 mM NaCl (Figure 1), the inhibitor of non-trypsin inhibiting proteins was removed by subsequent affinity chromatography on anhydrotrypsin-sepharose (Fiqura 2). The final purification was achieved by Mono SR cation exchange chromatography (Fiqura 3). The data of the isolation procedure are summarized in Table 1.

Table 1: Purification of the leech-derived tryptase inhibitor 3.5 grams of the lyophilized leech extract was used as the starting material. An inhibitory unit (IU) was defined as the amount of inhibitor that reduces the amidolytic activity of tryptase by 30 percent (see Methods). The isolated leech-derived tryptase inhibitor was homogeneous according to SDS-PAGE and N-terminal sequence analysis (Figures 4 and 8). However, two species were separated by reverse phase HPLC (Figure 5). Subsequent amino acid sequencing after tryptic fragmentation, amino acid analysis and mass spectroscopy (Table 2, Figures 6 and 7), demonstrated that the two species comprise three forms that differ only in their C-terminal sequence. Accordingly, the B (43 aa) and C (46 aa) forms differ from the shorter A form (42 aa) by a C-terminal extension of -GLY and -GLY-ILE-LEU-ASN, respectively. The results obtained for the three forms are compared in Table 3.

Table 2: Amino acid analysis of the two species of the leech-derived triptase inhibitor separated by HPLC (see Fiqura 5) The values given in parentheses are the values calculated from the amino acid sequence. -1) forms A and B have not separated; 2) The sequence of forms A and B contain 4 and 5 glycines, respectively; n.d. = not determined.

Table 3: Summary of the characterization of the three forms of the leech-derived tryptase inhibitor -1) Assuming three disulfide bonds 2) Forms A and B have not separated The 35 N-ter amino acid residues of the leech-derived inhibitor were determined by sequencing the native inhibitor. The primary structure was completed and verified using overlapping peptides generated after modification and tryptic and / or chymotryptic fragmentation (Figure 8). Sequence comparisons show that the leech-derived tryptase inhibitor is a non-classical Kazal-type serine proteinase inhibitor. The highest degree of similarity was found in Bdellin B [Frink Rehm, 1986]; In the common sequence section for both inhibitors (amino acids 1-40), 19 of 40 (47.5 percent) amino acids are identical (Table 4). Despite the high sequence identity for the leech-derived tryptase inhibitor, Bdellin B, an inhibitor also isolated from the medical leech, does not affect tryptase (unpublished observations).

Table 4: Comparison of the amino acid sequences of the tryptase inhibitor derived from leech and Bdellin B-3 [Fink Rehm, 1986].

Trip inhibitor 1 5 10 5 20 5 30 5 4) Bdellin rate 3 KKVCACPKILKPVCGSDGRTYANSCIARCNGVSIKSEGSC DTECVCTKELHRVCGSDGVTYDNECLATCHGASVAHDHAC .. *. *. * * .. ****** **. *. *. * *. *. *. . . * Inhibitor of trip-PT rate Bdellin 3 EGHEEHHVDEHGEDHD (* = identical residues; = homologous amino acids) Example 2: Specificity of the leech-derived inhibitor The leech-derived tryptase inhibitor inhibits human tryptase in a concentration-dependent manner. Using the tripeptide-nitroanilide of Tos-Gly-Pro-Arg-pNa as a substrate, a maximum inhibition of 50 percent was observed (Fiqura 9). Accordingly, more possibly due to steric hindrance, the inhibitor blocks only two of the four catalytic subunits of the tryptase tetramer, leaving the other two subunits accessible to the small substrate. The interaction of the inhibitor with the first two tryptase subunits can be mathematically described as a tight binding inhibition with a K1 of approximately 1.4 nM for the complex. The leech-derived tryptase inhibitor is highly specific, and inhibits only trypsin and chymotrypsin with affinities similar to those for tryptase (Table 5). In contrast, K¿ values for complexes with other proteinases are at least 200 times higher.

Table 5: Specificity of the leech-derived tryptase inhibitor, f1) in 0.2 μM; 2.) Ki for the inhibition of two of the four subunits of the tryptase tetramer; nor, no inhibition in 0.2 μM; nd, not determined) Table 5 (continued) Table 5 (continued) Example 3: Biological characterization To determine whether the leech-derived tryptase inhibitor affects the dissociation of a biologically relevant substrate by tryptase, its effect on the decomposition of the vasoactive intestinal peptide (VIP) was measured. At a concentration of 4 x 10 ~ 7M, the inhibitor reduced the decomposition of VIP by 66 percent (Fiqura 10). Accordingly, the inhibitor blocks not only the tryptase-induced dissociation of the nitroanilide substrate of the tos-Gly-Pro-Arg-pNA peptide (see Example 2), but also that of the biologically relevant substrate. Tryptase not only dissociates soluble proteins, but also directly interacts with cells that activate cellular functions, such as the growth of fibroblasts and keratinocytes. To determine whether the leech-derived tryptase inhibitor blocks these cellular effects of tryptase, its effect on the tryptase-induced growth of cultured human keratinocytes was studied. In the absence of the inhibitor, tryptase (10 ~ 9 M) markedly stimulated the growth of keratinocytes, increasing their incorporation of 3H-thymidine to 182 + 6 percent of the control. The leech-derived tryptase inhibitor did not significantly affect baseline growth, suggesting a lack of cytotoxic effects (Table 6).

Table 6: Effect of the leech-derived tryptase inhibitor on the proliferation of the human keratinocyte cell line HaCaT The growth rates were calculated as the incorporation of 3H-thymidine in the presence of the agonist and / or inhibitor expressed as a percentage of the incorporation in medium only. The data is given as the average + EM, n > 2. The inhibitor greatly reduces the cell growth induced by tryptase (10-9 M) without a significant effect on proliferation under baseline conditions. Accordingly, the inhibitor almost completely blocks the biological effect of tryptase without any cytotoxic side effects. However, the inhibitor significantly reduced the tryptase-induced proliferation, reducing the incorporation of 3H-thymidine to 115 + 5 percent and 120 + 2% of the control, at a concentration of 10 ~ 7 M and 10 ~ 8 M, respectively. Since this incorporation of 3H-thymidine is similar to that caused by 10-11M tryptase (118 + 4%), the data suggest that the inhibitor blocks the cellular effect of tryptase by approximately 99 percent. Finally, the influence of the leech-derived tryptase inhibitor on prothrombin time (according to Quick), and the partial thromboplastin time, to determine whether it interferes with blood coagulation was measured. At a concentration of 10 ~ 7M, the inhibitor had no significant effect on both parameters (Table 7). Accordingly, the leech-derived tryptase inhibitor does not significantly inhibit any of the proteases involved in the blood coagulation cascade.

Table 7: Effect of the leech-derived tryptase inhibitor on blood coagulation The inhibitor (100 nM) does not affect the prothrombin time according to Quick, and at the time of partial thromboplastin, demonstrating that the enzymes involved in the coagulation cascade are not inhibited.

Example 4: Pharmaceutical preparation containing the tryptase inhibitor for parenteral administration A solution prepared according to Example 1 is dialyzed against a NaCl solution of a concentration of 0.9 percent. The concentration of the solution is then adjusted to 1 milligram / milliliter or 10 milligrams / milliliter, by concentration or by dilution with the same NaCl solution. These solutions are sterilized by ultrafiltration (the pores membranes having 0.22 microns). Sterilized solutions can be used for intravenous administration.

Example 5: Preparation of recombinant tryptase inhibitor 5.1. Materials All the products used were obtained in Sigma, St. Louis, USA; Merck, Darmstadt, FRG; Serva, Heidelberg, FRG; Biomol, Hamburg, FRG; Roth, Karlsruhe, FRG, Braun, Melsungen, FRG; Dianova, Hamburg, FRG; Promega, Madison, USA. Restriction endonucleases and DNA modifying enzymes were purchased from Boehringer, Mannheim, FRG; New England Biolabs, Beverly, USA, and Pharmacia-Biotech, Freiburg, FRG. [35 S] -adthosine-5-triphosphate-a, was obtained from Amersham Buchler, Braunschweig, FRG. Bacto-tryptone, Bacto-peptone, Bacto yeast nitrogen base (without amino acids, W / O), Bacto yeast extract and Bacto-agar were from disk, Augsburg, FRG. As the culture medium we use 2xYT [Sambrook et al., 1989]; YPD (10 grams of Bacto yeast extract, 20 grams of Bacto-pectone, 20 grams of glucose, pH of 6.0); YED (20 grams of Bacto yeast extract, 20 grams of glucose, 6.7 grams of NaH2P04, pH of 6.0), and SD + (6.7 grams of Bacto base nitrogen (w / o), 20 grams of glucose, 6.7 grams of NaH2P04 , 19 milligrams of L-leucine, pH 6.0). Oligonucleotides were purchased from MWG-Biotech, München, FRG, or were synthesized by Dr. S. Modrow, München, FRG.

Vectors and strains: The cloning vector E. coli pUC, was from Pharmacia Biotech Europe GmbH, Freiburg. The launch and expression vector of E. coli - S. cerevisiae, pVT102U /, and yeast strain S-78, were kindly provided by T. Vernet, Montreal, CAN, and by C. -W. Chi and Y. -S. Zhang, both from Shanghai, China [Lit. Vernet et al., Chen et al.). E. coli TG1 ((lac-pro), supE, thi, hsdD5 / F * traD36, proA + B +, laclq, lacZmld) was from Amersham Buchler, Braunschweig, FRG; E. coli JM105 (thi, rspL, endA, sbcB15, hspR4, (lac-proAB) F'traAB proAB, laclq, lacZM15); and E. coli HB101 (F ", pro", leu ", trhi" lacY, Smr, endon ", recA", rk ~, mk ~) were from Deutsche Stam sammlung Braunschweig, FRG. Standard molecular cloning techniques were performed according to Sambrook et al [Sambrook et al., 1989], and with M.D. Rose et al [Rose et al., 1990]. 5.2. Standard analytical methods a) SDS PAGE and isoelectric focusing (IEF) SDS-PAGE of the proteins was performed with 15 to 25 percent polyacrylamide gels, following the procedure of Laemmii [Laemmii, 1970]. The gels were self-prepared and executed in a conventional apparatus, or in the PhastSystem (Pharmacia, Solentuna, Sweden). Isoelectric focusing was also done with the PhastSystem, using the isoelectric focusing calibration kit, pH 3 to 10. b) HPLC analysis, amino acid sequencing Normally 2 to 3 nanomoles of protein were analyzed by reverse phase HPLC, as detailed previously [Auerswald et al., 1991]. The N terms were sequenced with a 473A gas phase sequencer (Applied Biosystems GmbH, Witerstadt, FRG), following the manufacturer's instructions. c) Determination of protein concentrations To determine the protein concentration, Pierce BCA * Protein Assay was used, with BSA as the standard protein [Smith et al., 1985]. The A280nm (1%) was calculated for the recombinant LDTI-C, using the values of A280 for the aromatic residues and the cystines of Mach et al. [1992]: A280 (1%) = 3.46, and for the protein mixtures, A28o (i%) = 1 «d) Trypsin inhibition assay The concentration of the inhibitoryly active material and the specific inhibitory activity of rLDTI-C, was determined indirectly by measuring the residual trypsin activity using the following conditions described by Chase and Shaw, 1970. Test regulator: 0.05M Tris-HCl, pH 7.6, 150 mM NaCl. 0.1 percent (volume / volume) of Triton X-100, 600 pM trypsin; lOOμM of Tos-Gly-Pro-Arg-p-NA. e) Determination of Ki values The equilibrium dissociation constants (K ^) were determined for rLDTI-C complexes with trypsin and tryptase, essentially as described previously [Bieth, 1980]. 5.3.3. Construction of the synthetic LDTI-C gene A synthetic gene encoding a recombinant homolog of LDTI form C was designed and constructed, as illustrated in Figure 11. The DNA sequence was selected on the basis of the amino acid sequence of the inhibitor of LDTI. natural tryptase by means of the GCG sequence analysis software [UWGCG, Devereux et al., 1984], with the codon uses of E. coli and S. cerevisiae for strongly expressed genes [Bennetzen and Hall, 1982]. The 5'-OH ends of the internal oligonucleotides were phosphorylated using T4 polynucleotide kinase, prior to hybridization. The six oligonucleotides, each 200 picomoles, were heated for 5 minutes at 95 ° C. Hybridization was achieved during cooling to room temperature within 8 hours. After extraction with phenol and precipitation with ethanol, internal notches were ligated by T-4 ligase (Boehringer), in accordance with the manufacturers' protocol. The material was separated by gel electrophoresis on a low melting point agarose (5 percent), and a 149 bp long fragment was purified using the MERMAID isolation kit from Dianova, Hamburg. 5.4. Construction of the cloning vector pRM3.l.l? The DNA fragment obtained according to 5.3 was ligated into the vector pUCld cut with EcoRI / HindIII (molar ratio of the vector: fragment, 1: 20). The E cells. Competent TG1 coli were transformed with the ligation mixture, and recombinant clones were selected. DNA sequencing was performed using the sequencer M13 / pUC (-40), a 17 mer sequencer, and the reverse sequencer preparer (-48), a 24 mer. The pRM3.1.10 vector (Figure 15) containing the designed sequences of rLDTI-C was used for other experiments. 5.5 In vitro production and cytoplasmic expression in E.coli a) The synthetic LDTI-C gene and the pASK 40 expression vector [Skerra et al., 1991] were separately dissociated with EcoRI and HindIII, purified, and ligated. Modified pASK 40 was designated pRM4.1.4 (Figure 16). The DNA of pRM4.1.4 was analyzed by an in vitro transcription transfer system coupled with E. coli S-30, from Promega, with cysteine S-35, following the instructions of the manufacturers. The transfer of in vitro transcription of the vector pRM4.1.4 with a lysate of E. coli S-30, commercially available, showed two major radioactive labeled protein bands with an approximate molecular weight of 7 kDa and 5 kDa (data not shown). The other strong band detected appears to be β-lactamase (approximate molecular weight of 31 kDa). The 7 kDa protein band is interpreted as the undissociated fusion protein containing the signal sequence ompA and LDTI-C (theoretical molecular weight 7038 Da), while the protein band of 5 kDA (theoretical molecular weight of 5015 Da) appears to be the dissociated and expected LDTI-C [ANS], which is extended by three amino acid residues. b) For cytoplasmic expression, the synthetic LDTI-C gene was ligated after a filling reaction in pGEX-3X (Pharmacia) dissociated with Smal. The resulting vector was called pRM 11.1.4 (Figure 17), and the resulting host strain is E. coli 1314. Cytoplasmic fusion proteins of glutathione-S-transferase-LDTI-C were found as insoluble inclusion bodies, with E. coli 1314 (HB 101 with pRMll.1.4, data not shown). 5.6. Construction of the pRM expression vector 9.1.4 For the yeast expression experiments, the modified alpha coupling secretion system pVT102U / a was selected [Vernet et al., 1987], where the trypsin inhibitor Trichosan thes was successfully expressed , a small inhibitor of serine proteinase from the macerated family [Chen et al., 1992]. Within this system, the recombinant inhibitor was correctly doubled, dissociated from the signal sequence, protected from proteolytic degradation, and could be purified in two or three stages from the yeast fermentation broth. In order to use the launch vector pVT102U / a, first the rLDTI-C gene had to be modified. The LDTI-C gene (Figure 11) was used by replacing the Eco-RJ / Sphl cartridge with a Sbal / Sphl linker cartridge. The sequence of this Sbal / Sphl linker is CTAGATTAAAAGAAAGAAGGTTTGCGCATGV. It codes for the C-terminal end (Figure 11 c) of the alpha coupling type signal sequence containing a dissociation site for the signal peptidase KEX2 (Lys Arg) and N terminus of LDTI. The modified LDTI-C gene was assembled by ligation of three fragments using the Xbal / Sphl linker cartridge, the Sphl / HindIII LDTI-C fragment, and the Xbal / HindIII dissociated pUCld vector (molar ratio, 10: 5: 1) . After the transformation of TG1 E. coli, the recombinant clones were sorted by restriction analysis and DNA sequencing using the preparer M13 / pUC (-40) (Biolabs) and the reverse preparer M13 / pUC / -48 (Biolabs) (CGCAGTAGCGGTAAACG). The new vector pRM 5.1.5 (Figure 12a) carried the expected sequence and the Xbal / HindIII fragment, including the rLDTI-C gene was ligated into pVT102U / dissociated with Xbal / HindIII. The resulting expression vector pRM 9.1.4 (Figure 12b) was isolated and used to transform S. cerevisiae strain S-78, in accordance with the method of Becker and Guárante [Becker and Guárante, 1991]. 5.7. Expression in Saccharomyces cerevisiae Analytical rLDTI-C expression experiments were performed using yeast strain H005 (S-78 with pRM9.1.4), with Fernbach flasks (180-220 rpm, 28 ° C; Pre-culture for 3 days with 100 milliliters of SD medium (+), and main cultures for 4 days with 900 milliliters of fresh YED medium). On each day, the cell density was determined (OD700), the pH was adjusted to 6.0 with 1M NaOH, and 10 milliliters of a solution of 50 percent yeast extract material, and 30 milliliters of 50 glucose were added. percent (weight / volume), and trypsin inhibition was determined. After transformation of the competent S-78 strains with pRM9.1.4, the expression of rLDTI was detected. The broth of the cultured recombinant yeast cells showed remarkable inhibition of trypsin. The concentrated supernatant gave a protein pattern with the strongest band migrating at approximately a molecular weight of 5000 Da after SDS-PAGE (see Figure 13, lane 2). The recombinant material was isolated in preparation from the S culture broth. cerevisiae grown in one-liter agitator flasks for 96 hours. After this time, the culture curve of the yeast cells reached an OD700 of 22.0. The trypsin inhibitory activity was detected after two days, and increased parallel to the biomass. The yeast broth was harvested (6000 grams, 20 minutes, 4 ° C) after 96 hours of culture, and the supernatant was filtered, first through a 0.16 micron membrane, and then through a cross-flow membrane. with a cut of 3 kDa (Filtron Omega Minisette, Filtron, Karlstein, FRG). The regulator was exchanged by diafiltration with 20 mM NaH2P04, pH 8.2. The material was purified by cation exchange chromatography (Fractogel EMD S03 ~ 65o (S) column 150-10; Merk), flow rate of 3 milliliters / minute, elution buffer of 20 mM NaH2P04, pH of 8.2, 500 mM NaCl. The data of a representative purification are summarized in Table 8.

Table 8: Results of a typical purification of rLDTI form C, from culture supernatant of Saccharomyces cerevisiae The total protein was estimated by applying the Pierce assay (bovine serum albumin as standard); the active material was calculated from trypsin inhibitor assays; the yield is given as a percentage of the isolated material. From a liter of fermentation broth, 3 milligrams of rLDTI-C were obtained. The SDS-PAGE of this material showed a homogeneous but relatively broad band migrating at an approximate molecular weight of 5000 Da (Figure 13, lane 3). Approximately 85 percent of rLDTI-C was eluted as an acute peak in 28 percent acetonitrile, when analyzed by HPLC RP18. The amino acid sequencing of peak 1 (Figure 14) revealed the expected N-terminus KKVCACPK. But small heterogeneities were observed after RP HPLC, and a different N-terminus (peak 2) was identified, starting with an additional 11 amino acids from the C-terminal part of the alpha factor signal peptide (Figure 14). This demonstrates that the endogenous KEX 2 protease of the yeast was dissociated with high precision after LysArg, the recognition site of the KEX2 signal peptidase, and still opposite the two N-terminal amino acid residues Lys Lys of LDTI. The isoelectric approach with the PhastSystem showed that the isoelectric point of rLDTI was above a pH of 10. The inhibition constants determined from the tryptase-LDTI-C and trypsin-LDTI-C complexes are similar to those of natural LDTI. The measured specific trypsin inhibitory activity of 60 percent is comparable with other recombinant inhibitors. RLDTI-C inhibits human tryptase in a manner similar to the naturally occurring leech-derived tryptase inhibitor: using the tripeptide-nitroanilide of tos-Gly-Pro-Arg-pNa, as a substrate, maximum inhibition was observed. approximately 50 percent, and a Ki of 1.9 nM that was calculated for the complex between tryptase and rLDTI-C.

Example 6: Construction of pFBYl66 pFBY166 is a plasmid derived from pUC18 which contains a BamHI fragment of 1085bp. This fragment contains the CUP1 promoter fused with ARG from the factor a leader, a sausage fragment, and the factor a terminator. The precise manner in which the fusions were designed, makes possible the insertion of ORF (open reading frame) containing fragments either in ATG by using the EcoRI site, after the signal sequence by using a PsTI site, or after the leader sequence of factor a, by insertion after the BglII site. The ORF to be expressed should ideally have a SalI site at its 3 'end to facilitate fusion with the terminator, which is preceded by a SalI site, and should not have Ba HI sites in its sequence, since dissociation of this plasmid in the two eitioe BamHI eepara the entire expression cartridge, so that it can be easily cloned into a yeast release vector. PFBY166 contains a 425 bp Ba Hl / EcoRI fragment of the CUP1 promoter, corresponding to nucleotides 1080 to 1505 of the EMBL GENBANK accession number K02204. The CUP1 promoter allows expression in a copper-regulated manner. The ATG is provided as part of the sequence and pheromone signal leader of factor a-1, nucleotides 293 to 527 of the EMBL GENBANK accession number K01581, followed by the sequence, AGATCTTGC, which positions a BglII site, which is unique in pFBY139, just before the normal position for the KEX2 dissociation site of LysArg. If fusions are required for only one signal sequence, this can be achieved by using the unique PstJ site that is present within the region encoding the signal sequence. The BslII site is followed by an unimportant sequence, since it is always removed when the ORF of entry into the plasmid is cloned., between any of the EcoRI, PstJ, or BglII sites, and the Salí site, which marks the end of the sausage fragment, and the beginning of the pheromone factors of factor a-1, nucleotides 825 to 1100 of EMBL GENBANK access number X01581. This is followed immediately by the sequence AATTCGGATCC, which encodes the BamHI site that limits the end of the expiry cartridge. This plasmid can be constructed using polymerase chain reaction (PCR) fragments of the genomic DNA of the yeast. All oligonucleotides used in the PCR reaction are synthesized using an automatic DNA synthesizer. The PCR reactions are carried out in a Perkin Elmer PCR unit, under the following conditions: 20 mM of the oligonucleotides in question are incubated in 0.1 milliliter of regulator (10 mM Tris-HCl, pH 8.3, 50 mM KCl , 1.5 mM MgCl2) with 2.5 units of Taq DNA polymerase, and 0.2 mM of dATP, dCTP, dTTP, and dGTP. The reactions are incubated for 30 cycles: 30 seconds at 92 ° C, for 1 minute at 42 ° C and at 72 ° C for 1 minute. The fragment comprising most of the signal of factor A and the leading eequences, ee generates from the genomic yeast DNA using the fragments of PCR 1 and 2: 1. 5 'GTGCGAATTCAAAATGAGATTTCCTTCAATTTTTACTGCAG 3 • 2. 5' CAAAGTCGACTTTATCCAGCAAGATCTCTTCTTCTTTAGCAGCAATGC 3 ' fragment comprising the factor a terminator is generated from genomic yeast DNA using PCR fragments 3 and 4: 3. 5 'GAAGAGATCTTGCTGGATAAAGTCGACTTTGTTCCCACTGTACTTTTAGC 3' 4. 5 'CCGGGGATCCGAATTAATTCTCTTAGGATTCG 3' The fragment comprising the CUP1 promoter is generated from genomic yeast DNA using the 5 and 6: 5 PCR fragments. 5 'TAGAGGATCCCCATTACCGACATTTGGGCGCTATACGTGC 3 »6. 5' CGACGAATTCACAGTTTGTTTTTCTTAATATCTATTTCG 3 'and the subeaseous dissociation with BamHI and ecoRI. The fragment comprising most of the factor signal to and the leader sequences, and the fragment comprising the factor a terminator, are mixed and reamplified in a PCR reaction with oligonucleotide 1 and oligonucleotide 3, and cut with ecoRI and BamHI. The last amplified fragment and the fragment comprising the CUP1 promoter, are cloned into pTZ18R cut with BamHI, and treated with bacterial alkaline phosphataea, to create pFBY139.

Example 7: Construction of pHE 174 Expression of low tryptase inhibitor in control of the regulated CUP1 promoter An etyretic gene encoding the tryptase inhibitor in the use of the preferred yeast codon is assembled from 3 synthetic oligonucleotides in a reaction of PCR In addition, the gene extends at its end 51 to provide a fusion within the convenient frame with the alpha factor leader in plasmid pFBY 166. The following 3 oligonucleotides are synthesized using an automatic DNA synthesizer: 1. 5 • -AAAGATCTTG CTGGATAAAA GAAAGAAGGT TTGCGCCTGT CCAAAAGATTT TGAAGCCAGT TTGTGGTTCT GACGGTCGTA CC-3 '2. 5'-ACAAGAACT TCAGACTTAA TAGAAACACC GTTACAACGG GCAATACAAG ACTTGGCGTA GGTACGACCG TCAGAACCAC-3' 3. 5 • -TTGTCGACTC AGTTCAAAAT ACCGGTTGGA CAAGAACCTT CAGACTTAA 3 'of these 3 oligonucleotides, a 170bp fragment is assembled in the next reaction of polymerase chain (PCR), using the Perkin Elmer PCR unit, and the following conditions: 20 mM of oligonucleotides 1 and 3, and 20 mM of oligonucleotide 2, are incubated in incubated in 0.1 milliliter of regulator (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2) with 2.5 units of Taq DNA polymerase, and 0.2 M of dATP, dCTP, dTTP, and dGTP. The reaction is incubated for 30 cycles: 30 seconds at 92 ° C, for 1 minute at 42 ° C and at 72 ° C for 1 minute. The 170 bp PCR fragment is isolated on a 2 percent agarose gel, restricted with BglII and Scill and ligated into pFBY 166 (supra), cut into BglII and SalI. The e . coli HB101 is transformed with the resulting plasmid pHE174. The strain of E. Transformed coli is designated as E. col i / pHE174. The correct fusion of the PCR fragment with the leader of factor a and the correct sequence of the ORF of the tryptase inhibitor is confirmed by sequencing.

Example 8: Construction of pHE 175 and 175R 2-micron vectors, with the expression cartridge of the tryptase inhibitor. For expression in yeast, pDP34 is used as vector. PDP34 (EP-A-340 170, Figure 3 thereof) is an E-yeast release vector. coli, with the ampicillin resistance marker for E. coli and the selective yeast markers of URA3 and dLEU2. It contains the sequences of 2 full micras in form A, and is efficient in REP1, REP2 and FLP. Plasmid pDP34 is digested with BamHI, and sticky ends are desfoephorylated by treatment with alkaline phosphatase. PHE174 is digested with BamHI, and the 1119bp fragment containing the complete tryptase inhibitor expression cartridge bound in pDP 34 cut in BamHI. The E coli HB 101 is transformed with the resulting plasmids pHE 175 and 175R. The orientation of the insert is tested by digestion with Sali. PHE 175 contains the tryptase inhibitor expression cartridge in a clockwise orientation with respect to dLEU2; the pHE 175R in the counter-clockwise orientation with respect to the dLEU2 marker.

EXAMPLE 9 Construction of pHE 176 The ORF of the Tryptase Inhibitor fused with the invertase signal sequence (SUC2) to provide a seventh alternative secretion, the ORF of the triptaea ee inhibitor fuses with the signal sequence of the yeast invertase gene SUC2. The following 2 oligonucleotides are made: 1. 5 '-TTGTCGACTC AGTTCAAAAT A-3' 2. 5 '-AAGAATTCAT GCTTTTGCAA GCTTTCCTTT TCCTTTTGGC TGGTTTTGCA GCCAAAATAT CTGCAAAGAA GGTTTGCGCC TGTC-3' pHE 174 is used as the template DNA for a chain reaction of polymerase as described in Example 7. 20 nanograms of the template pHE 174 are incubated with 20 mM of the oligonucleotide preparation under the experimental conditions as in Example 7. The amplified PCR fragment of 214 bp is isolated on a gel. of agarose at 2 percent, restricted with EcoRI and Salí, and ligated into the vector pFBY 166 cut in EcoRI and Salí. The E coli HB 101 is transformed with the resulting plasmid pHE 176. The correct sequence of the tryptase inhibitor fusion of the SUC2 signal sequence is confirmed by sequencing.

Example 10: Construction of pHE 177 and pHE 177R 2-micron vectors with the tryptase inhibitor expression cartridge with the SUC2 signal sequence In analogy to Example 8, the BamHI fragment of 918 bp containing the expression cartridge of the tryptase inhibitor, is separated from pHE 176 by digestion with BamHI, and inserted into pDP 34 cut in BamHI. The E coli HB 101 is transformed with the resulting plasmids pHE 177 and pHE 177R.

The orientation of the insert is tested by digestion with I left. The pHE 177 contains the tryptase inhibitor expression cartridge in a clockwise orientation with respect to dLEU2, and the pHE 177R in a counterclockwise orientation.

Example 11: Construction of Saccharomyces cerevisiae strain TR 1456 The Saccharomyces cerevisiae strain TR1456 is constructed as described in EP-A-341 215. Starting with Saccharomyces cerevisiae strain H449 (DMS 4413, MATa, leu23, 112, ura3, prbl °]), in two subsequent series of experiments, the two carboxypeptidaeae yeca and yscY are removed, from strain H449, by alteration of their coding genes KEX1 and PRC1, respectively. First the gene encoding ysca, KEX1, is altered. For this purpose, the H449 strain is transformed with a DNA fragment encoding the KEXI gene, with the complete URA3 gene inserted in the middle of the coding region of KEX1. Prototropic uracil transformants are selected and tested for the absence of ysca activity. Next, the URA3 gene inserted in the KEXI site is altered by transformation with a plasmid containing an altered version of the gene, URA3Δ5 (see EP-A-341 215). Transformants that are autotrophic for uracil are selected, and in the next step they are altered in their endogenous PRC1 gene that codes for yscY. of carboxypeptidase. The experiment is carried out in a completely analogous manner, as described for the alteration of KEX1. The finally reegenic isogenic derivative of strain H449 is called TR1456, and has the following genotype: TR1456 = MATa, leu2-3, 112, ura-3, prbl, kexl :: ura3, prcl :: ura3, [cir °] Example 12: Transformation of strain TR 1456 with plasmids pHE 175, 175R, 177 and 177R Plasmids pHE 175, 175R, 177 and 177R are introduced into host strains H449 and TR1456, respectively, using the traneformation protocol defined by Hinnen and colaboradoree (Proc. Natll, Acad. Sci. USA (1978), 75, 1929).

Other details of the procedure are as described in EP-A-341 215. The trane-formed yeast cells are selected on minimal yeast medium, supplemented with leucine, and lacking uracil. The simple transformed yeast clones are isolated and are referred to as: Saccharoceae cerevisiae TR 1456 / pHE 175 Saccharomyces cerevisiae TR 1456 / pHE 175R Saccharomyces cerevieiae TR 1456 / pHE 177 Saccharomyces cerevisiae TR 1456 / pHE 177R Saccharomyces cerevieiae H449 / pHE 175 Saccharomycee cerevieiae H449 / pHE 175R Saccharomycee cerevieiae H449 / pHE 177 Saccharomycee cerevisiae H449 / pHE 177R Example 13: Secretion of leech-derived tryptase inhibitor by TR 1456 transformed with plasmid pHE 177 Saccharomyces cerevisiae TR 1456 / pHE 177 cells are cultured in two subsequent precultures of 20 milliliters each. The synthetic medium consists of: 6.7 grams / liter of Difco yeast nitrogen base (without amino acids) 10 grams / liter of L-aeparagine 1 gram / liter of L-hietidine 20 grams / liter glucose 0.02 grams / liter L-leucine The pH of the medium is adjusted to 5.8. The first preculture is grown for 60 hours at 28 ° C and 180 r.p.m. The second preculture is inoculated with 2 percent (volume / volume) of the first preculture, and incubated for 24 hours at 28 ° C and 180 r.p.m. The main culture medium consists of: 5 grams / liter of peptone 10 grams / liter of yeast extract 20 grams / liter of glucose 40 grams / liter of sucrose 3 grams / liter of ammonium sulfate 2 grams / liter of dihydrogen phosphate potaeio 0.5 grams / liter of magnesium sulfate etahydrate 0.1 grams / liter of sodium chloride 0.1 grams / liter of calcium chloride 10 ~ 5 grams / liter of biotin The main culture (100 milliliters of medium) is inoculated with approximately 106 cells /milliliter, and incubated for 72 hours at 28 ° C and 180 r.p.m. Immediately after inoculation, sterile copper sulfate at a concentration of 1 mM is added to the culture. At the end of the fermentation, aliquots of the culture are taken, the cells are removed by centrifugation, and the culture supernatant is analyzed by the activity of the leech-derived tryptase inhibitor, by titration of the inhibitor with trypsin, as described in 2.2. . i).

Example 14: Analysis of the leech-derived tryptase inhibitor from fermentation cultures of Saccharomyces cerevisiae strain TR 1456 / pHE177, using reverse phase HPLC Samples of the culture supernatants of strain TR 1456 / pHE177, are subjected to analysis HPLC under the following conditions: A Merck Lichrospher 1000 RP-8 column (4 x 250 millimeters, 10 microns) is used. The mobile phase A is made from water (Nanopur®, Barnstead) containing 0.1 percent (volume / volume) of trifluoroacetic acid. The mobile fae B is made from 20 percent water (NanopurR, Barnstead) and 80 percent acetonitrile (HPLC grade, Fluka), containing 0.09 percent (volume / volume) of trifluoroacetic acid. The chromatographic separations are performed at a flow rate of 1.5 milliliters / minute, with the following gradient running (Table 9). The eluents are monitored by absorbance at 214 nanometers.

Table 9 A higher pH is observed with a retention time of 19.5 minutes on the chromatogram that is present in the strains carrying the inhibitor expression plasmid, but absent in the non-transformed strains. Another analysis revealed that this peak contains a leech-derived tryptase inhibitor species with an apparent Mr of 4738.8, as detected by mass spectroscopy. This value is in agreement with the calculated Mr value of 4738, indicating the inhibiting molecule of the entire length that leads to both the N term and the correct C term. The chemical molecular weight of the inhibitor is determined by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) using a homemade instrument (Boernsen et al., Chimica (1990) 44, 412-416).

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LIST OF SEQUENCE (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: UCP Gen-Pharma AG (B) STREET: Kraftstr. 6 (C) CITY: Zuerich (E) COUNTRY: Switzerland (F) ZIP CODE: CH-8044 (G) TELEPHONE: 01 251 10 60 (ii) TITLE OF THE INVENTION: Triptase Inhibitor (iii) NUMBER OF SEQUENCES: 9 (iv) LEGIBLE COMPUTER FORM: (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Paentln Rellease # 1.0 , Version # 1.2 (EPO) (2) INFORMATION FOR THE SEQUENCE IDENTIFICATION NUMBER: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: No (vi) ORIGINAL SOURCE: (A) ORGANISM: Hirudo medicinalis (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER D SEQUENCE: 1: Lys Lys Val Cys Ala Cys Pro Lye lie Leu Lys Pro Val Cys Gly Ser 1 5 10 15 Asp Gly Arg Thr Tyr Wing Asn Ser Cys lie Wing Arg Cys Asn Gly Val 20 25 30 Ser lie Lys Ser Glu Gly Ser Cys Pro Thr 35 40 (2) INFORMATION FOR THE IDENTIFICATION NUMBER OF SEQUENCE: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Hirudo medicinalis (iii) HYPOTHETIC: No (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER D SEQUENCE: 2: Lys Lys Val Cys Ala Cys Pro Lys lie Leu Lys Pro Val Cys Gly Ser 1 5 10 15 Asp Gly Arg Thr Tyr Wing Asn Ser Cys lie Wing Arg Cys Asn Gly Val 20 25 30 Ser lie Lys Ser Glu Gly Ser Cys Pro Thr Gly 35 40 (2) INFORMATION FOR IDENTIFICATION NUMBER SEQUENCE FICTION: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: No (xi) DESCRIPTION SEQUENCE: IDENTIFICATION NUMBER D SEQUENCE: 3: Lys Lys Val Cys Ala Cys Pro Lys lie Leu Lys Pro Val Cys Gly Ser 1 5 10 15 Asp Gly Arg Thr Tyr Wing Aen Ser Cye lie Wing Arg Cye Asn Gly Val 20 25 30 Ser lie Lys Ser Glu Gly Ser Cys Pro Thr Gly lie Leu Asn 35 40 45 (2) INFORMATION FOR THE SEQUENCE IDENTIFICATION NUMBER: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 126 base pairs (B) TYPE: nucleic acid (C) CORDS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: YES (iii) COUNTER-MEANING: NO (vi) SOURCE ORIGINAL: (A) ORGANISM: Hirudo medicinalis (Xi) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER SEQUENCE: 4: AARAARGTNT GYGCNTGYCC NAARATHYTN AARCCNGTNT GYGGNWSNGA YGGNMGNACN TAYGCNAAYW SNTGYATH GC NMGNTGYAAY GGNGTNWSNA THAARWSNGA RGGNWSNTGY CCNACN (2) INFORMATION FOR THE SEQUENCE IDENTIFICATION NUMBER: 5 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 129 base pairs (B) TYPE: nucleic acid (C) CORDS: single (D) ) TOPOLOGY: linear (Ü) TYPE OF MOLECULE: cDNA (Üi) HYPOTHETICAL: YES (iii) COUNTER-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Hirudo medicinalis (Xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NUMBER : 5: AARAARGTNT GYGCNTGYCC NAARATHYTN AARCCNGTNT GYGGNWSNGA YGGNMGNACNSNTGYATHGC NMGNTGYAAY GGNGTNWSNA THAARWSNGA RGGNWSNTGY CCNACNGGN (2) INFORMATION FOR THE SEQUENCE IDENTIFICATION NUMBER: 6 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 138 base pairs (B) TYPE: nucleic acid (C) CORDS: single (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (Üi) HYPOTHETICAL: YES (iii) CONTRA-SENTÍDO: NO (Vi) ORIGINAL SOURCE: (A) ORGANISM: Hirudo medicinalis (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER SEQUENCE: 6: AARAARGTNT GYGCNTGYCC NAARATHYTN AARCCNGTNT GYGGNWSNGA YGGNMGNACN TAYGCNAAYW SNTGYATHGC NMGNTGYAAY GGNGTNWSNA THAARWSNGA RGGNWSNTGY CCNACNGGNA THYTNAAY (2) INFORMATION FOR THE SEQUENCE IDENTIFICATION NUMBER: 7 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 149 paree of baee (B) TYPE: nucleic acid (C) CORDS: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: SI ( iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Hirudo medicinalis (xi) DESCRIPTION OF SEQUENCE: IDENTIFICATION NUMBER SEQUENCE: 7: ATTTCGAAGA AGGTTTGCGC ATGCCAAAG ATCTTGAAGC CAGTCTGTGG TTCTGACGGT 6 CGTACATATG CTAACTCATG CATCGCTCGT TGTAACGGTG TATCGATCAA GTCTAAGGT 12 TCTTGTCCAA CCGGTATTTT AAACTAATA 14 (2) INFORMATION FOR THE SEQUENCE IDENTIFICATION NUMBER: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 149 base pairs (B) TYPE: nucleic acid (C) CORDS: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: YES (iii) COUNTER-SENSE: YES (vi) ORIGINAL SOURCE: (A) ORGANISM: Hirudo medicinalis (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER SEQUENCE: 8: AGCTTATTAG TTTAAGTTAC CGGTTGGACA AGAACCTTCA GACTTGATCG ATACACCGTT 6 ACAACGAGCG ATGCATGAGT TAGCATATGT ACGACCGTCA GAACCACAGA CTGGCTTCAA 12 GATCTTTGGG CATGCGCAAA CCTTCTTCG 14 (2) INFORMATION FOR THE SEQUENCE IDENTIFICATION NUMBER: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 929 base pairs (B) TYPE: nucleic acid (C) CORDS: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 435..440 (D) OTHER INFORMATION: / function = "EcoRI site" ( ix) FEATURES: (A) NAME / KEY: miec_feature (B) LOCATION: 642..647 (D) OTHER INFORMATION: / function = "eitio Salí" (ix) FEATURES: (A) NAME / KEY: developer (B) LOCATION: 1.443 (D) OTHER INFORMATION: / phenotype = "CUP1 promoter" (ix) CHARACTERISTICS: (A) NAME / KEY: sig_peptide (B) LOCATION: 444..500 (D) OTHER INFORMATION: / function = "SUC2 invertase signal sequence" (ix) CHARACTERISTICS: (A) NAME / KEY: matjpeptide (B) LOCATION: 501..641 (D) OTHER INFORMATION: / product = "tryptase inhibitor" (ix) FEATURES : (A) NAME / KEY: terminator (B) LOCATION: 648.-923 (D) OTHER INFORMATION: / standard_name = "alpha factor termination" (ix) FEATURES: (A) NAME / KEY: misc_feature (B) LOCATION : 924..929 (D) OTHER INFORMATION: / function = "BamHI site" (Xi) DESCRIPTION OF SEQUENCE: IDENTIFICATION NUMBER SEQUENCE: 9: GATCCCCATT ACCGACATTT GGGCGCTATA CGTGCATATG TTCATGTATG TATCTGTATT TAAAACACTT TTGTATTATT TTTCCTCATA TATGTGTATA GGTTTATACG G-ATGATTTAA 1 TTATTACTTC ACCACCCTTT ATTTCAGGCT CATATCTTAG CCTTGTTACT AGTTAGAAAA 1 AGACATTTTT GCTGTCAGTC ACTGTCAAGA CATTCTTTTG CTGGCATTTC TTCTAGAAGC 2 AAAAAGAGCG ATGCGTCTTT TCCGCTGAAC CGTTCCAGCA AAAAAGACTA CCAACGCAAT 3 ATGGATTGTC AGAATCATAT AAAAGAGAAG CAAATAACTC CTTGTCTTGT ATCAATTGCA 3 TTATAATATC TTCTTGTTAG TGCAATATCA TATAGAAGTC ATCGAAATAG ATATTAAGAA 4 AAACAAACTG TAACGAATTC AAAATGCTTT TGCAAGCTTT CCTTTTCCTT TTGGCTGGTT 4 TTGCAGCCAA AATATCTGCA AAGAAGGTTT GCGCCTGTCC AAAGATTTTG AAGCCAGTTT 5 GTGGTTCTGA CGGTCGTACC TACGCCAACT CTTGTATTGC CCGTTGTAAC GGTGTTTCTA 6 TTAAGTCTGA AGGTTCTTGT CCAACCGGTA TTTTGAACTG AGTCGACTTT GTTCCCACTG 6 TACTTTTAGC TCGTACAAAA TACAATATAC TTTTCATTTC TCCGTAAACA ACATGTTTTC 7 CCATGTAATA TCCTTTTCTA TTTTTCGTTC CGTTACCAAC TTTACACATA CTTTATATAG_7_CTATTCACTT CTATACACTA AAAAACTAAG ACAATTTTAA TTTTGCTGCC TGCCATATTT 8 CAATTTGTTA TAAATTCCTA TAATTTATCC TATTAGTAGC TAAAAAAAGA TGAATGTGAA 9 TCGAATCCTA AGAGAATTAA TTCGATCC 9

Claims (37)

NOVELTY OF THE INVENTION Having described the above invention, it is considered as a novelty, and therefore, the content of the following is claimed as property: CLAIMS

1. A purified human tryptase inhibitor molecule, which is a polypeptide; or functional equivalents thereof that show an inhibitory activity of triptaea.

2. The tryptase inhibitor according to claim 1, which can be obtained from extracts of leeches.

3. The tryptase inhibitor according to claim 1 or 2, characterized by: a) inhibiting human tryptase with a K value on the scale of about 0.1 to 10 nM; and b) leaving the proteinases involved in the human blood coagulation cascade substantially unaffected.

4. The tryptase inhibitor according to claim 1 in claim 1, characterized essentially by the amino acid sequence: Lys-Lys-Val-Cys-Ala-Cys-Pro-Lys-Ile-Leu 10 Lys- Pro-Val-Cys-Gly-Ser-Asp-Gly-Arg-Thr 20 Tyr-Ala-Asn-Ser-Cys-Ile-Ala-Arg-Cys-Asn 30 Gly-Val-Ser-Ile-Lys-Ser- Glu-Gly-Ser-Cys 40 Pro-Thr-X 42 wherein the C-terminal residue X represents H (SEQUENCE IDENTIFICATION NU: 1), -Gly (SEQUENCE IDENTIFICATION NUMBER: 2), or -Gly-Ile-Leu-Asn (SEQUENCE IDENTIFICATION NUMBER: 3); or functional equivalents thereof, having one or more of the amino acids of the above sequence substituted or deleted, or having one or more amino acids added, without substantially affecting its tryptase inhibitory activity.

5. A functional equivalent of the inhibitor according to claim 1 of claim 1, which comprises the amino acid sequence: R1 -Cye-Pro-Lye-Ile-Leu Lys-Pro-Val-Z-Gly- Ser-Asp-Gly-Arg-Thr Tyr-Ala-Asn-Ser-Cys-Ile-Ala-R2 wherein: the N-terminal residue R1 represents Ala- or Cys-Ala-; the C-terminal residue R2 represents -Arg or -Arg-Cys; and Z defines any amino acid.

6. An inhibitor according to claim 1 in any of claims 1 to 5, which can be obtained by peptide synthesis or recombinant DNA technology.

7. A polynucleotide that encodes a polypeptide with a tryptase inhibitory activity, according to claim as claimed in any of claims 1 to 6; or the complementary polynucleotide thereof.

8. The polynucleotide in accordance with claim 7 claimed in, characterized in that includes nucleotide sequence (SEQUENCE IDENTIFICATION NUMBER: 4): 1 AARAARGTNTGYGCNTGYCCNAARATHYTNAARCCNGTNTGYGGNWSNGA 51 YGGNMGNACNTAYGCNAAYWSNTGYATHGCNMGNTGYAAYGGNGTNWSNA 101 THAARWSNGARGGNWSNTGYCCNACNX wherein R denotes A or G; M denotes A or C; W denotes A or T; S denotes C or G; And denotes C or T; H denotes A, C, or T; N denotes any nucleotide; X denotes 3 '-OH (SEQUENCE IDENTIFICATION NUMBER: 4) GGN (SEQUENCE IDENTIFICATION NUMBER: 5) or GGN ATH YTN AAY (SEQUENCE IDENTIFICATION NUMBER: 6) or the complementary cord thereof; and the nucleotide sequences that hybridize in the aforementioned DNA sequence.

9. The polynucleotide according to claim 7, characterized in that-, includes a nucleotide sequence that substantially corresponds to the nucleotide residues 1 to 149, or preferably 7 to 144, of the SEQUENCE IDENTIFICATION NUMBER:; or a fragment of it.

10. The polynucleotide according to claim 7, characterized in that it includes a nucleotide sequence that corresponds substantially to the nucleotide residues 1 to 149, or preferably 10 to 147 of the SEQUENCE IDENTIFICATION NUMBER: 8; or a fragment of it.

11. An oligonucleotide that hybridizes to a nucleotide sequence that encodes a polypeptide with a tryptase inhibitory activity.

12. The oligonucleotide according to claim as claimed in claim 11, characterized in that it includes a nucleotide sequence that is substantially complementary to the nucleotide sequence of residues 22 to 87 of the SEQUENCE IDENTIFICATION NUMBER: 5.

13. A polynucleotide that encodes a polypeptide with a tryptase inhibitory activity, and that is can be obtained by hybridization with an oligonucleotide according to claim 1 in one of claims 11 and 12.

14. A polypeptide expression cartridge comprising a promoter operably linked to a DNA sequence encoding the polypeptide in accordance with claim one of claims 7 to 10 and 13, and with a DNA sequence containing transcription termination signals.

15. The expression cartridge according to claim 14, characterized in that it includes a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the appropriate reading frame, with a second polypeptide encoding a DNA sequence according to claim 1 in one of claims 7 to 10 and 13, and a DNA sequence containing tranectication termination signal.

16. The expression cartridge according to claim 15, characterized in that the promoter is selected from the group consisting of CUPlp, and GAPDHp.

17. The expression cartridge according to claim 14 or claim 15, characterized in that the signal sequence is selected from the group consisting of the leader of factor a, PH05, and SUC2.

18. The expression cartridge according to claim one of claims 14 to 17, characterized in that the terminator is selected from the group consisting of the factor terminator a and the PH05 terminator.

19. The expression cartridge according to claim 18, characterized in that it includes the DNA sequence of the SEQUENCE IDENTIFICATION NUMBER: 9, or a functional equivalent of the element.

20. A polypeptide encoded by a polynucleotide according to claim 13.

21. A vector for the transformation of eukaryotic or prokaryotic hosts, which comprises a polynucleotide according to claim as claimed in one of claims 7 to 10 or 13.

22. A vector for the transformation of eukaryotic or prokaryotic hosts, which comprises an expression cartridge in accordance with the claim in one of claims 14 to 19.

23. The vector according to claim 21 or 22, characterized in that it is a yeast vector based on two microns.

24. The vector according to claim one of claims 21 to 23, selected from: a) pRM9.1.4, as deposited with the DSM, and having access number DSM 9271; b) pRMll.1.4 as deposited with the DSM, and having the access number DSM 9272; c) pRM5.1.5 as deposited with the DSM, and having the access number DSM 9270; d) pRM4.1.4 as deposited with the DSM, and having the access number DSM 9269; e) pRM3.1.10 as deposited with the DSM, and having the accession number DSM 9268.

25. The vector according to claim as claimed in one of claims 21 to 23, selected from the group consisting of pHE175, pHE175R, pHE177, and pHE177R.

26. A method for the preparation of a human tryptase inhibitor, by: a) obtaining a leech extract, and b) purifying the extract by means of dialysis and column chromatography.

27. The method according to claim 26, characterized in that: a) the extract is dialyzed against a suitable regulator; b) the dialysed extract is purified by cation exchange chromatography; biospecific affinity chromatography, and an additional step of cation exchange chromatography.

28. A method for the preparation of a recombinant tryptase inhibitor, which method includes: a) transforming a prokaryotic or eukaryotic host with a vector according to claim 1 of claims 21 to 25; b) inducing the expression of the coding sequence of the tryptase inhibitor; c) recovering the expression product; and optionally d) removing from the obtained product, fragments of the peptide not required for the tryptase inhibitory activity, and / or optionally renaturing the product.

29. A tryptase inhibitor that can be obtained by a method according to claim 28.

30. A pharmaceutical composition that includes a tryptase inhibitory amount of a polypeptide according to claim 1 of any of claims 1 to 6, 20 and 29, optionally in combination with a pharmaceutically acceptable carrier or diluent.

31. The use of a tryptase inhibitor according to claim 1 in any of claims 1 to 6, 20 and 29, in the diagnosis of functional disorders.

32. The use of a polypeptide according to claim 1 in any of claims 1 to 6, 20 and 29, for the preparation of a pharmaceutical composition for the treatment of disorders related to mast cells.

33. Use according to claim 31, for the preparation of a pharmaceutical composition for the treatment of asthma, interstitial lung disease, arthritis, periodontal disease, allergic disorders, skin disorders, psoriasis, or disorders of blood clotting

34. A prokaryotic or eukaryotic host transformed with a vector according to claim 1 of claim 21 to 25., and variants and mutants thereof.

35. A host according to claim 34, characterized in that it is Saccharomyces cerevisiae or E. Coli.

36. A eukaryotic host according to claim claimed in claim 35, deposited with the DSM, and having accession number DSM 9273, and variants and -nutants thereof capable of producing a tryptase inhibitor polypeptide.

37. A host according to claim 35, which is selected from the group consisting of: Saccharomyces cerevisiae TR 1456 / pHE 175 Saccharomyces cerevisiae TR 1456 / pHE 175R Saccharomyces cerevisiae TR 1456 / pHE 177 Saccharomyces cerevisiae TR 1456 / pHE 177R Saccharomyces cerevisiae H449 / pHE 175 Saccharomyces cerevisiae H449 / pHE 175R Saccharomyces cerevisiae H449 / pHE 177 Saccharomyces cerevisiae H449 / pHE 177R, and variants and mutants thereof, capable of producing a tryptase inhibitor polypeptide. In testimony of which, I sign the above in this City of Mexico, D. F., on the 25th day of the month of July 1994. BY UCP GEN-PHARMA AG APPOINED Ing. Javier Saucedo C.