HK1006183B - Expression and secretion vectors for hirudine by way of transformed yeasts - Google Patents

Description

The present invention relates to vectors for the expression of the DNA sequence coding for hirudin or hirudin analogues, the secretion of hirudin in the culture medium of yeasts transformed by these vectors and processes for the fermentation of active hirudin and the resulting hirudin.

The anticoagulant activity found in the salivary glands of the medicinal leech, Hirudo medicinalis, comes from a small polypeptide called hirudin (1). This highly specific and highly effective thrombin inhibitor has been extensively studied in recent times as it has the potential to be a very interesting therapeutic agent. However, the extreme difficulty and cost of isolating and purifying it has prevented it from being used more widely, or even being studied clinically.

The possibility of producing hirudin by cloning genes and their expression by recombinant DNA has already been demonstrated by the cloning of a natural leech gene coding for hirudin and expression in the E. coli microorganism (French patent application No 84 04755 on behalf of the applicant filed on 27 March 1984). Although a peptide with biological activity has been produced in E. coli, it is very important to produce hirudin in other types of microorganisms.

In addition, the hirudin synthesized by E. coli remains intracellular and therefore must be purified from a very large number of E. coli peptides. For these reasons, it was interesting to express the hirudin gene in yeast which does not produce pyrogenic or toxic substances for humans and which is able to secrete proteins in the culture medium.

The mechanism of action of hirudin as an anticoagulant is only beginning to be understood. The substrate for binding hirudin is thrombin, which is a proteolytic enzyme, which by activation (by activated factor X) from its zymogenic form, prothrombin, cuts fibrinogen in the circulatory stream to convert it into fibrin which is necessary for blood clot formation. The thrombin-hirudin complex dissociation constant 1:1 (0.8 x 10−10) indicates an extremely strong association between these molecules.[2] Practically, the non-covalent complex between these two molecules can be considered inseparable in vivo.

Hyrudin is a very specific thrombin inhibitor, with a much higher affinity than the natural substrate fibrinogen. In addition, it is not necessary to have other clotting factors or other plasma constituents. Hyrudin's specific and very important anti-thrombin activity makes its clinical application as an anticoagulant evident.

Hirudine has been studied extensively in animals for its anticoagulant properties. The most detailed study (3) describes the activity of hirudine in the prevention of venous thrombosis, vascular occlusion and disseminated intravascular coagulation (DIC) in rats. Hirudine is well tolerated in rats, dogs, rabbits and mice when in highly purified form and injected intravenously. The DL50 in mice is greater than 500 000 U/kg body weight (i.e. 60 mg/kg). Another study (4) indicates that mice tolerate doses up to 1 g/kg twice a week and that repeated intravenous and intravenous injections of up to 10 mg/kg do not lead to acute sensitization in mice.

In addition, hirudin in the experimental animal is rapidly eliminated (half-life of about 1 hour) still in biologically active form via the kidneys (3).

Two other independent studies, one using dogs (5) and the other (6) demonstrating the activity of hirudin in the prevention of CID in rats, are in agreement with the positive results of Markwardt and colleagues, who recently published the first in vivo analysis of the effects of natural hirudin on the human hemostatic system (7).

It has also been shown that hirudin prevents endotoxin-induced DIC in pigs (8) and thus represents a potential solution to the very serious problems posed by endotoxinemia which lead to high mortality in pigs.

Intravenously administered hirudin has a half-life of 50 minutes and 50% of the active form of hirudin is found in the urine for 24 hours after injection. An increase in clotting time (measured in vitro for thrombin, thrombophroplastin and prothrombin) is observed depending on the plasma concentration of hirudin, showing that the molecule maintains its biological activity in the circulation for 2 weeks; no signs of acute reactivity have been observed in the urine, as in the case of fibrinogen injections, no significant changes in the level of active substances in the system, and no signs of secondary sensitization have been observed.

These studies suggest that hirudin may be an interesting clinical agent as an anticoagulant. The pre-clotting phase of blood is not affected due to the high specificity of hirudin's action. The anti-thrombin activity is dose-dependent and the effect of hirudin is rapidly reversible due to its rapid renal elimination. Hirudin has been shown to be much superior to heparin for the treatment of CID (3, 6) as might be expected given that CID is accompanied by a decrease in anti-thrombin III (a co-factor necessary for the action of heparin) and a co-regulation of platelet factor 4 which is a very effective antiparetin agent.

One study has highlighted the possibility that hirudin can be absorbed by human skin (10), although the results obtained remain somewhat difficult to interpret.

Commercial preparations of raw acellular extracts of leeches are available in ointment form (Hirucreme, Société Nicholas, France; Exhirud-Blutgel, Plantorgan Werke, RFA), but further testing with higher doses of highly purified material is needed to establish whether this is an interesting route of administration. Generally, the preferred routes of administration are intravenous, intramuscular and percutaneous. Other routes of administration have been reported for hirudin, notably oral (BSM No. 3 792 M).

This product may also be used in combination with other ingredients to treat psoriasis and other skin disorders of the same type as described in DOS 2 101 393.

Hyrudin can also be used as an anticoagulant in clinical laboratory tests and as a research tool, in which case the high specificity for a single step in blood clotting may present a considerable advantage over the most commonly used anticoagulants, which are much less specific in their action.

In addition, hirudin may be very useful as an anticoagulant agent in extracorporeal circuits and dialysis systems where it may have considerable advantages over other anticoagulants, particularly if it can be immobilized in active form on the surface of these artificial circulatory systems.

The binding activity of hirudin to thrombin may also allow indirect protection of clotting factors such as factor VIII during its purification.

Finally, the use of labelled hirudine can be a simple and effective method of measuring thrombin and prothrombin levels. In particular, labelled hirudine can be used to visualize clots in formation because the clotting phenomenon involves the conversion of circulating prothrombin to thrombin at the site of formation, with the labelled hirudine attached to the thrombin and able to be visualized.

It is also possible to provide for the direct use of processed yeasts as a hirudin-releasing medicinal product, for example by spreading a cream containing these yeasts which secrete hirudin on the skin.

In summary, the hirudin of the invention has a wide range of possible applications: (ii) as an anticoagulant in critical thrombotic conditions for prophylaxis and prevention of the spread of existing thrombosis; (iii) as an anticoagulant to reduce hematomas and swelling after microsurgery, situations where a large use of live leeches is made; (iv) as an anticoagulant in extracorporeal circulation systems and as an anticoagulant to coat synthetic biomaterials; (iv) as an anticoagulant in clinical tests of blood samples in laboratory tests; (v) as an anticoagulant in clinical research on clotting and as a topical agent for the treatment of varied blood disorders; (vi) as a topical agent for the treatment of hemorrhoids, edema and other blood disorders; (vi) as a preservative, and (vi) as a preparation for the treatment of edema and other blood disorders; (vi) as a preservative, and (vi) as a preservative, may be used in the treatment of edema and other blood disorders; (vi) as a preparation for the treatment of edema and other blood disorders; (vi) as a preservative, and (vi) as a preservative of psoriatin; (vi) as a preservative; (vi) as a preservative; (vi) as a preservative; (vi) as a preservative; (vi) as a preservative; (vi) as a preservative; (vi) as a preservative; (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi) (vi

As an indication, hirudin may be used in therapeutic formulations at concentrations corresponding to 100-50 000 anti-thrombin U/kg/day.

Since hirudin is water-soluble, it is easy to obtain injectable or other-applicable pharmaceutical formulations using pharmaceutically acceptable media and vehicles.

Finally, hirudin marked either by radioactive marking or by any other type of enzymatic or fluorescent marking by known techniques may be used for in vitro measurements or in vivo imaging, in particular for visualization of clot formation.

A preparation of hirudin from the whole animal was used to determine the amino acid sequence of the protein (11,12).In subsequent experiments, a gene that expresses itself as messenger RNA was cloned in the heads of the fasting leeches.This gene carries information for a protein (hirudin variant 2 or HV-2) whose sequence is significantly different from that found throughout the animal's body (called variant protein called HV-1).There are 9 differences in amino acid residues between HV-1 and HV-2 and the differences between the two NH2-terminal residues (val-val or ileth-thr) may explain the apparent contradictions in the literature regarding the end of the NH2-hirudin (13).

Figure 1 shows the DNA sequences of the recombinant plasmid pTG717 containing a copy of the cDNA corresponding to the mRNA of HV-2, as well as the amino acid sequence derived from the DNA sequence, in b), and the differences between this sequence and the amino acid sequence of HV-1, in c).

A variant of the HV1 hirudin is described in FEBS LETTERS, 1984, Vol. 105 (2), pages 180-184.

It should be noted that the cDNA sequence is probably not complete and that a signal sequence may exist upstream of the start of the mature protein.

The expression of cDNA of HPV-2 in microorganisms shows that the corresponding protein has antithrombin activity.

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One of the objects of the present invention is the preparation of hirudin by yeast.

Yeasts are single-celled eukaryotic organisms.The genus Saccharomyces includes strains whose biochemistry and genetics are studied intensively in the laboratory; it also includes strains used in the food industry (bread, alcoholic beverages, etc.) and therefore produced in very large quantities.

The ease with which the genetics of Saccharomyces cerevisiae cells can be manipulated, either by conventional techniques or by techniques derived from genetic engineering, and even better by a combination of these two types of techniques, and the long industrial history of this species make it a preferred host for the production of foreign polypeptides.

EP-A-123 294 describes yeast transformation vectors that secrete hybrid precursor polypeptides to recover the desired polypeptide.

EP-A-116 201 describes vectors of secretion in yeasts using the alpha-leading sequence.

EP-A-129 073 describes the expression of GRF in yeasts using, inter alia, elements from the alpha factor gene.

EP-A-123 544 describes protein secretion using elements from alpha factor genes.

Therefore, the present invention relates in particular to a functional DNA block for the preparation of hirudin from yeast, characterised by at least: . the hirudin gene or one of its variants (hereinafter H gene) ;. a DNA sequence (Str) containing the signals for the transcription of the H gene by yeast.

This functional block, which is embedded in a plasmid or in the chromosomes of a yeast, preferably of the genus Saccharomyces, may allow, after transformation of that yeast, the expression of hirudin, either in active form or as an inactive precursor, capable of regenerating hirudin by activation.

The interest of Saccharomyces cerevisiae is that it is able to secrete some proteins in the culture medium, the study of the mechanisms responsible for this secretion is in full swing, and it has been shown that it is possible to make the yeast secrete, after adhoc manipulation, properly processed human hormones similar in all respects to those found in human serum (14, 15).

In the present invention, this property is used to obtain the secretion of hirudin because this has many advantages.

First, yeast secretes little protein, which has the advantage of enabling, if the secretion of a given foreign protein is managed at a high level, to obtain in the culture medium a product which may represent a high percentage of the total protein secreted, and thus facilitate the purification of the protein sought.

There are several proteins or polypeptides secreted by yeast, and in all known cases these proteins are synthesized as a longer precursor whose NH2-terminal sequence is decisive for entry into the metabolic pathway leading to secretion.

The synthesis in yeast of hybrid proteins containing the NH2-terminal sequence of one of these precursors followed by the sequence of the foreign protein may, in some cases, result in the secretion of this foreign protein. The fact that this foreign protein is synthesized as a precursor, and therefore generally inactive, allows the cell to be protected from the potential toxic effects of the target molecule, the cleavage which releases the active protein only taking place in vesicles from the Golgi apparatus which isolate the protein from the cytoplasm.

The use of metabolic pathways leading to secretion to produce a foreign protein in yeast therefore has several advantages: (i) it allows a reasonably pure product to be recovered from the culture medium; (ii) it protects the cell against the possible toxic effects of the mature protein.3

Therefore, the expression blocks according to the invention will have in particular the following structure: - Str-Lex-Scl-H-gene - What is it? Lex code for a leader sequence needed to the excretion of the protein corresponding to the H gene; Scl is a DNA sequence coding for a cleavage site; in addition, the Scl-gene H element can be repeated several times.

The alpha pheromone system was chosen as an example of a secretion system, i.e. in the previous sequence, the Lex sequence is derived from the yeast alpha sex pheromone gene, but other systems could be used (e.g. the Killer protein system) (14).

Yeast sex pheromone α is a 13-amino acid peptide (framed in Figure 2) that is secreted in the culture medium by Matα-type S. cerevisiae yeasts. The factor α stops opposite-sex type cells (Mata) in the G1 phase and induces biochemical and morphological changes necessary for the mating of the two cell types. Kurjan and Herskowitz (17) cloned the structural gene for factor α and deduced from the sequence of this gene that this 13-amino acid factor α was synthesized as a 165-amino acid precursor pre-protein (Figure 2).Err1:Expecting ',' delimiter: line 1 column 306 (char 305)

The nucleotide sequence of this precursor also contains 4 HindIII restriction sites indicated by an H arrow.

Several fusions between the pheromone α gene and the mature sequence of hirudin have been achieved. Matα-type yeast cells can express these fused genes. The corresponding hybrid proteins can then be processed by the signals they contain, which come from the pre-pro sequences of the pheromone α precursor. Polypeptides with the hirudin sequence are therefore expected to be recovered from the culture surgeon.

In one construct, the Scl sequence has an ATG codon at the 3' end preceding the H gene; the fused protein therefore contains a methionine immediately upstream of the first amino acid in the mature hirudin sequence.

In other constructions, the cutting signals normally used to produce the pheromone α are used to produce polypeptides with anti-thrombin activity in the culture surgeon. This is the case when the Scl sequence has at its 3' end two codons coding for Lys-Arg, i.e. -AAA or AAG with AGA or AGG; the polypeptide is cut by an endopeptidase which cuts into COOH and Lys-Arg dipeptides, releasing hirudin.

In particular, the invention relates to constructions in which the sequence predating the hirudin gene encodes for one of the following amino acid sequences: 1) Lys Arg Glu Ala Glu Ala Trp Leu Gln Val Asp Gly Ser Met Hirudine ...,2) Lys Arg Glu Ala Glu Ala Hirudine ...,3) Lys Arg Glu Ala Glu Ala Lys Lys Arg Hirudine ...4) Lys Arg Glu Ala Glu Ser Leu Asp Tyr Lys Arg Hirudine ... or5) Lys Hir Arg...

It is of course possible to envisage using other sequences which, at the amino acid level, are selectively cut by an enzyme, provided that this cleavage site is not also present in the hirudin itself.

Finally, the expression blocks will be able to have a yeast terminator sequence, for example the PGK gene, after the H gene.

In general, the expression blocks of the invention can be incorporated into a yeast, in particular Saccharomyces, either in an autoreplicating plasmid or in the yeast chromosome.

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The present invention also relates to yeast strains transformed by a block of expression according to the invention, either carried by a plasmid or incorporated into its chromosomes.

When the promoter is the pheromone α gene, the yeast will preferably be of the Matα sex type. For example, a strain of genotype ura3− or leu2− or other, complemented by the plasmid, will be used to ensure that the plasmid is maintained in the yeast by appropriate selection pressure.

Although it is possible to prepare hirudin by fermentation of previously processed strains on an appropriate culture medium, by accumulation of hirudin in cells, it is nevertheless preferable, as shown in the previous description, to secrete hirudin in the medium either in mature form or as a precursor which will need to be processed in vitro.

This maturation can be done in several steps. First, it may be necessary to cleavage some elements from the translation of the Lex sequence, this cleavage will be done at the sequence level corresponding to Scl. As noted earlier, the mature hirudin can be preceded by a methionine which will be selectively cut by cyanogen bromide. This process is usable because the coding sequence of the hirudin does not include methionine.

It is also possible to predict at the N-terminus the Lys-Arg dipeptide which is cut into COOH by a specific endopeptidase; this enzyme being active in the secretion process, the mature protein can thus be obtained directly from the medium.

In some cases, particularly after treatment with cyanogen bromide, it may be necessary to renature the protein by recreating the disulfide bridges, by denaturing the peptide, for example with guanidinium hydrochloride, and then renaturing it in the presence of reduced and oxidized glutathione.

Finally, the invention relates to hirudin obtained by the processes according to the invention.

Further features and advantages of the present invention will be shown by reading the following examples.

The figures in the annexes: Figure 1 shows the nucleotide sequence of the cloned cDNA fragment of the hirudine in pTG 717;Figure 2 shows the nucleotide sequence of the sex pheromone precursor α;Figure 3 shows the structure of pTG834;Figure 4 shows the structure of pTG880;Figure 5 shows the structure of pTG882;Figure 6 shows the structure of M13TG882;Figure 7 shows the structure of pTG874;Figure 8 shows the structure of pTG876;Figure 9 shows the structure of pTG0510;Figure 15 shows the structure of pTG834;Figure 4 shows the structure of pTG880;Figure 5 shows the structure of pTG882;Figure 5 shows the structure of pTG882;Figure 6 shows the structure of M13TG882;Figure 8 shows the structure of pTG885;Figure 9 shows the structure of pTG10510;Figure 15 shows the structure of pTG1888;Figure 11 shows the structure of pTG886;Figure 14 shows the structure of pTG1888;Figure 12 shows the structure of pTG1889;Figure 12 shows the structure of pTG1889;Figure 12 shows the structure of pTG1889;Figure 12 shows the structure of pTG1889;Pigure 12 shows the structure of pTG1889;Tis obtained after the transformation of the different proteins in the pTG1888;Pg8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8G8

Amino acid and nucleotide sequences are not included in this description to avoid burdening it but are explicitly included.

Example 1 - Construction of pTG822

A fragment of the cDNA of hirudin HV-2 was extracted from a gel after digestion of the plasmid pTG717 with PstI and then digested by both HinfI and AhaIII enzymes. The HinfI enzyme cuts downstream from the first codon of the mature sequence of hirudin HV-2. The AhaIII enzyme cuts about 30 base pairs (3') behind the stop codon of the hirudin sequence. The resulting HinfI-AhaIII fragment was isolated on gelose and then electrolytically removed from this gelose.

The coding sequence for the natural protein was introduced into the vector pTG880. The vector pTG880 is a derivative of the vector pTG838 (Figure 3). The plasmid pTG838 is identical to pTG833 except for the BglII site near the transcription terminator of the PGK. This site was removed by the Klenow polymerase filler action to give pTG838.

The basic elements of this vector (Figure 3) are: the URA3 gene as a selection marker in yeast, the replication origin of the yeast plasmid 2, the ampicillin resistance gene and the replication origin of the E. coli plasmid pBR322 (the latter two elements allowing the propagation and selection of this plasmid in the coliform), the 5' region of the yeast PGK gene with the sequence of this gene on the side of the site, the PBR2I-PIII fragment and the replication origin of the PBR322 gene in the Salvation Army (these two latter elements allow the propagation and selection of this plasmid in the coliform), the PGK gene in 5' region with the sequence of this gene up to the site, the PBR2I-PIII fragment and the PBR322 gene terminator This application was already filed in the French D. No. 84 of May 1984.

The plasmid pTG880 was constructed from pTG838 (Figure 4) by inserting a short polylinker region (from a bacteriophage M13) into pTG838 cut by EcoRI and BglII, allowing a series of cloning sites to be located, in the order: BglII, PstI, HindIII, BamHI, SmaI and EcoRI, immediately after the 5' region flanking the yeast PGK gene. The DNA of the plasmid pTG880 was digested with BII and SmaI, and the fragment of this digestion was isolated and selected from a gel site. The HI/Ahirin fragment of pTG717, which contains the majority of the coding sequence of the vector, was then recovered from the same site and transformed into a translocation sequence of H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H2H

This plasmid was used to transform yeast cells to produce hirudin under the control of the PGK promoter. However, no hirudin activity was detected in the raw extracts of the cells transformed with this vector. The reason for the lack of active hirudin production is not yet clear. However, the pTG882 construction served as a source of hirudin coding sequence for the yeast secretion vectors described below.

Example 2 - Construction of pTG886 and pTG897

First, the BglII-EcoRI fragment (230 bp) of pTG882 containing the hirudin sequence was transferred to bacteriophage M13mp8 (Figure 6) between BamHI and EcoRI sites, resulting in the M13TG882 phage from which an EcoRI-HindIII fragment (approximately 245 bp) could be isolated. This fragment contains the entire HV-2 hirudin coding sequence, the BamHI/BII fusion site and the coagulant ends (Hind-EcoRI) allowing cloning in the yeast secretion vector pTG881 (Figure 9).

The plasmid pTG881 (10 kb) is an E. coli/yeast shuttle plasmid, replicating autonomously in both E. coli and Saccharomyces cerevisiae, uvarum and carlbergensis strains.

The introduction of this plasmid into E. coli results in resistance to ampicillin (and other β-lactam-type antibiotics). In addition, this plasmid carries the yeast LEU2 and URA3 genes that are expressed in E. coli and Saccharomyces strains. Therefore, the presence of this plasmid in E. coli or Saccharomyces results in complementation of strains deficient in β-isopropylmalate dehydrogenase or OMP decarboxylase.

The plasmid pTG881 is constructed as follows: The starting plasmid is pTG848 (identical to pTG849 described in French patent No 83 15716 except for the reversed orientation ura3 gene), and consists of the following DNA fragments (Figure 7): 1°) The approximately 3.3 kb EcoRI-HindIII fragment from the plasmid pJDB207 (18). The HindIII site corresponds to the coordinate 105 of the plasmid 2form B, the EcoRI site to the coordinate 2243. In this fragment is the LEU2 gene inserted by extension polydeoxyadenate/polydeoxythymidilate polydeoxygen in the PstI site of the 2form B fragment (18.2°) The HindIII fragment of the URA3 gene (19).3°) The large EcoRI (coordinate 0) -SalI (coordinate 650) fragment of pBR32 PG. In the PII site of this fragment was inserted the EcoRI-HindIII fragment (the ends of which were pre-extruded by the action of the KKKK gene in the presence of 4KKK nucleotides) and the PRI-PRI segment corresponds to the end of the PBR220 (20.52 pBR25) PRI-SalI gene (20.4KK) at the end of the PBR220 pBR2 (20).

The two fragments are bound and, before transformation, this binding mixture is subjected to the action of HindIII, which allows any form of plasmid that has retained one (or two) site HindIII to be eliminated. The E. coli strain BJ5183 (pyrF) is transformed and the transformed are selected for ampicillin resistance and for the characteristic place+. The result is the pTG874 pyramid (Figure 7) where the two sites are removed, thus giving the orientation of the URA3 gene to the same orientation in the transcription of the phospholipase (PGK).

The ends of the linear plasmid are made free by action of Klenow in the presence of the 4 nucleotides. Digestion by the SalI enzyme is then performed and the EcoRI (free end) -SalI fragment corresponding to the MFα1 gene is isolated. This latter fragment is bound to the SmaI-SalI (8,6 kb) piece of pGT874 to give the plasmid pGT876 (Figure 8).

In order to remove the proximal BglII site from the MFα1 promoter sequence, partial BglII digestion of the pTG876 plasmid was followed by Klenow action in the presence of the 4 deoxyribonucleotides. The new plasmid obtained, pTG881 (Figure 9), allows the insertion of foreign coding sequences between the first HindIII site of the MFα1 gene and the BglII site of the end of the PGK gene.

When fusion at site HindIII of the foreign coding DNA results in translation in the same reading phase, the resulting hybrid protein comprises the pre-pro portions of the α pheromone.

Cloning in pTG881 of the HindIII-EcoRI fragment carrying the hirudin gene leads to the plasmid pTG886 (Figure 10). Once cloned, a BglII site is found downstream of the fragment, which allows the fragment to emerge as HinfI-BglII, the HinfI site being the same as that used above. BII being unique in pTG881, it is very easy to reconstruct the 5' end of the hirudin coding sequence using 3 oligonucleotides reconstituting the 5' sequence of hirudin and allowing the pheromone α-gl protein sequence reading in the next phase of the mature hirudin sequence.

This new plasmid is called pTG897 (Figure 11).

Example 3 - Expression of hirudin by yeasts

The pTG897 DNA was used to convert a TGY1sp4 yeast (Matα, ura3 - 251 - 373 - 328, his3 - 11-15) into ura+ using a technique already described (21).

A processed colony was transplanted and used for the seeding of 10 ml of minimum medium plus casamino acids (0.5%). After 20 hours of culture, the cells were centrifuged, the surfactant dialysed against distilled water and concentrated by evaporation (vacuum centrifugation).

In parallel, a culture of TGY1sp4 transformed by pTG886 and a culture of TGY1sp4 transformed by a plasmid not sequencing for hirudin (TGY1sp4 pTG856) were treated in the same way. Dried cocoons were collected in 50 l of water and 20 l were boiled in the presence of 2.8% SDS and 100 mM mercaptoethanol deposited on an acrylamide-SDS gel (15% acrylamide; 0.1% SDS) (22). After fixation and colouring in Coosmassie blue, polypeptides can be expected to accumulate in the cystine of TGY14/TGY1sp6 and TGY1sp84/TGY7 and these peptides are very absent from the culture (see Figure 10).

The electrophoresis in Figures 12 and 13 was carried out as follows:

For Figure 12, the extracts were prepared as follows: 10 ml of minimum medium (Yeast Nitrogen Base Difco) without amino acid (6.7 g/l) glucose (10 g/l) supplemented with 0.5% casamino acids, were sown with different strains and cultured for 20 hours (stationary phase). The cells were centrifuged, the surfactant dialysed against water (minimum retention: PM 1000) and then dried by vacuum centrifugation. The samples were then collected with 50 l of load buffer, 20 of which are treated as described above and deposited on aclamide-Slamryde gel (15 % acryde, 0.1% Slamryde) (22).

The strains used are: . well 2 : TGY1sp4 transformed by a plasmid not containing the hirudin sequence (control); . well 3 : TGY1sp4 transformed by pTG886; . well 4 : TGY1sp4 transformed by pTG897; . well 1 : in well 1 the reference markers have been deposited (LMW Kit de Pharmacia; top to bottom: 94 000, 67 000, 43 000, 30 000, 20 100, 14 000).

The stripes are revealed by the blue colouring of Coomassie R-250.

After 10 minutes, the cells are centrifuged, taken up into 10 ml of complete medium (30°C) and incubated at 30°C under agitation. After 3 hours, the 10 ml of surfactant is dialysed against water and concentrated to a volume of 0.5 ml as described for Figure 12. Approximately 35 cpm (40l) deposited on SDS-acrylamide gel (15% SDS-acrylamide, 0.1% SDS-fluoride) are visualized.

The strains used are: . well 2 : TGY1sp4 transformed by a plasmid not containing the hirudin sequence;. well 3 : TGY1sp4 transformed by pTG886; . well 4 : TGY1sp4 transformed by pTG897; . well 1 : markers with PMs indicated (x103) have been deposited in well 1.

However, when supernatants containing these polypeptides were tested for anti-thrombin activity, no activity could be detected.

In the case of the polypeptide excreted by TGY1sp4 pTG886, it is expected to undergo the normal pheromone α precursor cleavage steps, which would result in an elongated 8-amino acid hirudin molecule at its NH2 end.

However, in the case of the polypeptide secreted by TGY1sp4/pTG897, the polypeptide is expected to be identical to the natural protein and therefore active. 1°) The protein is not fully matured, there may be residues of Glu-Ala in NH2 as described for EGF (16).2°) The protein is not active because it is not properly conformed or there are protein surfactants or PM 1000 molecules in the culture medium which inhibit activity.3°) The protein is not complete because it has undergone intracellular and/or extracellular proteolysis.

Example 4 - Activation by cutting with cyanogen bromide

If the cause of the lack of activity is related to the presence of additional amino acids in the NH2-terminal, it should be possible to restore activity by cleaving the peptide secreted by TGY1sp4/pTG886 by cyanogen bromide. This is because this reagent is specific to methionine residues and the fused protein encoded by pTG886 contains only one methionine. This reaction was carried out as follows: yeasts containing either the plasmid pTG886 or a control plasmid not containing a hirudine insert are cultured in 10 ml of total bromide. During this time, the culture reaches a density of 7−10.7 ml. The culture is then sold with a solution of 70 mg/ml of fresh bromide. The preparation for the analysis is prepared in the medium of 70 ml of concentrated dihydroxylic acid, which is then sold in the solution of 70 mg/ml of fresh bromide.

After removal of oxygen by a stream of nitrogen, the tubes are incubated in the dark for 4 hours at room temperature. All handling in the presence of cyanogen bromide is carried out with appropriate care and in a ventilated hood. The cleavage reaction by cyanogen bromide is stopped by adding 10 volumes of distilled water and then the solution is lyophilized.

The split peptides are dissolved in 10 ml of distilled water, and then re-lyophilized twice. Finally, the peptides are dissolved in a small volume of distilled water and an aliquot is used to measure antithrombin activity. The remainder of the sample is lyophilized and subjected to the renaturation steps described below.

Since the activity of hirudin depends on the presence of disulfide bridges in the molecule (1), it seemed likely that the cyanogen bromide cleaved peptide would need to be properly renatured to exhibit biological activity.

In summary, the freeze-dried peptides are dissolved in 400 l of guanidinium hydrochloride (GuHCl) 5 M in Tris HCl 250 mM, pH 9,0; then the solution is made into 2 mM reduced glutathione and 0,2 mM oxidised glutathione, in a final volume of 2.0 ml (final concentration is 1.0 M GuHCl and 50 mM Tris).

After 16 hours of incubation at 23°C in the dark, the samples are dialysed for 24 hours against 3 times 2 l of Tris HCl 50 mM, pH 7.5, NaCl 50 mM, at 23°C, and the final dialysate is clarified by centrifugation.

The result of this experiment, shown in Table I, clearly shows a recovery of antithrombin activity in the superswimming cells of cells infected with the pTG886 plasmid whereas there is no activity with the control plasmid. TABLEAU I

ACTIVITE ANTITHROMBINE DES SURNAGEANTS DE CULTURES DE LEVURES
Plasmide	Traitement du surnageant	Activité U/ml	Activité spécifique U/mg de protéines initiales
pTG886	a)après clivage avant renaturation	< 0,3	-
	b)après clivage et renaturation	2,46	102,5
contrôle	a)après clivage avant renaturation	< 0,3	-
	b)après clivage et renaturation	< 0,15	< 5,6

In conclusion, yeast cells carrying a recombinant plasmid can secrete a peptide with the biological activity of hirudin after cleavage and renaturation, indicating that the presence of additional amino acids in the NH2-terminal would be sufficient to explain the absence of activity of polypeptides in TGY1sp4/pTG886 cultures and possibly also in TGY1sp4/pTG897 cultures (Glu-Ala tails).

Example 5 - Introduction of a new cutting site just before the first amino acid of the coding sequence of hirudin HV-2 - plasmid pTG1805

In the pTG897 construction, there is a risk that the secreted peptide will retain Glu-Ala tails in NH2, which would explain the absence of antithrombin activity of this material. If this hypothesis is true, it should be possible to recover activity directly in the supernatant by creating a cutting site between the Glu-Ala residues and the first amino acid of the hirudin HV-2 (isoleucine). This was achieved by adding a coding sequence for the LysArg double, which is the recognition site for the endopeptidase involved in pheromone receptor maturation (Figure 2). The construction was obtained exactly as described for the plasma pTG897, in its plasma TGY14 (TgY14). The material was previously obtained by transmutation with the plasma pTG894 (TgY1844) and was used to analyze the plasma as a plasma.

The strains used are: . well 1: identical markers as in Figure 12; well 2: TGY1sp4 transformed by pTG897; well 3: TGY1sp4 transformed by pTG1805; well 4: control not producing hirudin.

The TGY1sp4/pTG1805 specific polypeptides migrate more slowly than the TGY1sp4/pTG897 specific polypeptides. This result suggests that the new cleavage site is not used effectively by the corresponding endopeptidase. Nevertheless, the biologically active superswimmer dosing of hirudin reveals that a small fraction of this material is active, unlike what is obtained with the clearly inactive TGY1sp4/pTG897 cultures (Table II). - What? TABLEAU II

ACTIVITE ANTITHROMBINE DES SURNAGEANTS DE CULTURES DE LEVURES (10 ml)
Plasmide	Activité spécifique U/mg	Activité totale U (10 ml)
pTG856	non détectable	-
pTG897	non détectable	-
pTG1805	21	2,0

Example 6 - New construction involving a new cutting site more efficient in releasing mature hirudin

In the previous buildup (pTG897), little hirudin activity was obtained in the supernatant, probably because the second cleavage site for the release of mature hirudin is poorly recognized, so a new buildup was made to make this additional cleavage site more susceptible to endopeptidase by adding a sequence of the 3 amino acids Ser Leu Asp upstream which is naturally present upstream of the first LysArg double (Figures 2 and 16).

The technique used is the same as described above except for the oligonucleotides whose sequences are reported below: The amino acid sequence in the cleavage region is shown in Figure 13. The corresponding plasmid is called pTG1818 and differs from pTG1805 only by the insertion of the 5'-TTG GAT AAA nucleotides corresponding to the Ser-Leu-Asp codons.

The activity dosed in the TGY1sp4 pTG1818 culture surfactants under the standard conditions described above is about 200 units per 10 ml of culture, or about 100 times higher than in the previous example.

Example 7 - Construction leading to synthesis of a precursor without Glu-Ala-Glu-Ala sequences

In the two previous examples, it was shown that the addition of a new Lys Arg site just upstream of the start of the mature sequence of hirudin allowed the release of active material into the supernatant. However, in these examples, if the precursor cut is done at the first Lys Arg site (distal to the start of the mature sequence), a heavier, inactive contaminant corresponding to elongated hirudin chains at the NH2 end can be obtained in the culture medium. This is particularly clear in the case of TGY1sp4 pTG1805 cultures where the inactive contaminant is predominant. This remains also plausible in the case of TGY1sp4 pTG18 cultures where the precursor is not predominantly active, but may be present in the case of TGY1 and GG891 Alaspyrin. To avoid the synthesis of this amino acid sequence, it is decided that the synthesis of the newly mature material should proceed by a fluidic process (1GFG894 and Alaspyrin).

The following strains were deposited at the National Collection of Microorganism Cultures (CNCM) of the Institut Pasteur, 28 rue du Docteur-Roux, 15ème Paris, on 30 April 1985: . TGY1sp4 pTG1818 : Saccharomyces cerevisiae, strain TGY1sp4 (Matα ura3-251-373-328-his 3-11-15) converted to ura+ by a plasmid pTG1818; deposit No I 441.

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Claims (21)

1. Yeast transformed by a functional DNA block integrated in a plasmid containing an origin of replication in yeast, or integrated in a chromosome of the said yeast, characterized in that the said block contains at least:

   - a DNA sequence coding for hirudin, one of its variants or a precursor of these (H gene),

   - a leader sequence (L_ex) containing the elements required for obtaining excretion of the gene product,

   - a DNA sequence (S_tr) containing the signals which provide for transcription of the H gene by yeast.
2. Transformed yeast according to Claim 1, characterized in that the said functional block contains at least the following sequences, from the 5' end to the 3' end:

   - a sequence (S_tr) containing the signals which provide for transcription of the H gene by yeast,

   - a leader sequence (L_ex) containing the elements required for obtaining excretion of the gene product,

   - the gene coding for hirudin, one of its variants or a precursor of these,

   - the gene coding for hirudin or one of its variants or precursors of these, preceded by a cleavage site S_cl.
3. Yeast according to either of Claims 1 and 2, characterized in that the sequence L_ex is that of the alpha sex pheromone of yeast.
4. Yeast according to one of Claims 1 to 3, characterized in that the leader sequence contains the pre fragment of the alpha sex pheromone of yeast.
5. Yeast according to one of Claims 1 to 4, characterized in that the block contains the prepro sequence of the alpha sex pheromone of yeast at the 5' end of the H gene.
6. Yeast according to one of Claims 1 to 5, characterized in that the H gene is followed by a yeast terminator sequence.
7. Yeast according to Claim 6, characterized in that the yeast terminator sequence is that of the PGK gene.
8. Yeast according to one of Claims 1 to 7, characterized in that the cleavage site is the dipeptide Lys-Arg.
9. Yeast according to one of Claims 1 to 7, characterized in that the functional block is integrated in a plasmid containing at least one origin of replication in yeasts.
10. Yeast according to Claim 9, characterized in that the origin of replication is that of the 2» plasmid.
11. Yeast according to one of Claims 9 and 10, characterized in that the plasmid contains, in addition, a selectable character.
12. Yeast according to Claim 11, characterized in that the selectable character is given by the URA3 gene.
13. Yeast according to one of Claims 1 to 12, characterized in that it is a yeast of the genus Saccharomyces.
14. Yeast according to Claim 13, characterized in that it possesses the Matalpha mating type.
15. Yeast according to one of Claims 12 to 14, characterized in that it is a strain of S. cerevisiae.
16. Process for preparing hirudin by fermentation of a yeast according to one of Claims 1 to 15 in a culture medium and recovery of the hirudin produced in the culture medium.
17. Process for preparing hirudin by fermentation of a yeast, characterized in that:

    a) a yeast strain containing a functional DNA block carried by a plasmid or integrated in a chromosome of the said yeast is cultured in a culture medium, characterized in that the DNA block contains at least the sequence:    ---S_tr---L_ex---S_cl---H gene---ter

    · S_tr is a DNA sequence containing the signals which provide for transcription of the H gene by yeast,

    · L_ex is a leader sequence enabling the excretion of hirudin, mature or in the form of a hirudin precursor which can be matured in vitro, to be obtained,

    · S_cl is a DNA signal coding for a cleavage site chosen from ATG and the sequences coding for Lys-Arg;

    b) the hirudin produced is recovered in the culture medium in mature form or in the form of a hirudin precursor which can be matured in vitro.
18. Process according to Claim 17, characterized in that the sequence S_cl-H gene contains a sequence coding for the sequence Lys-Arg-Ile-Thr.
19. Extracorporeal blood circuit, characterized in that at least part of the circuit in contact with the blood possesses a neutral surface of hirudin obtained by a process according to one of Claims 16 to 18.
20. Anticoagulant composition, characterized in that it contains yeasts according to one of Claims 1 to 15 as an agent which liberates hirudin.
21. Composition for visualizing the formation of a blood clot, characterized in that it consists of labelled hirudin obtained by a process according to one of Claims 16 to 18.