FI104635B - Hirudin producing transformed yeast and process for producing hirudin with this - Google Patents

Abstract

Functional block of DNA enabling to prepare hirudine from yeast, characterized in that it comprises at least: - the gene of hirudine or one of its variants (gene H); - A DNA sequence (Str) comprising the signals providing for the transcription of the gene H by the yeast.

Description

, 104635

Transformed yeast producing hirudin and a process for making hirudin therefrom.

The present invention relates to a yeast transformed with a functional DNA fragment integrated into a plasmid containing the origin of replication in the yeast or integrated into the chromosome of said yeast. Further described are expression vectors for DNA sequences encoding hirudin, secretion of hirudin produced by these vectors into the culture medium of transformed yeast, and a process for preparing active hirudin by fermentation, as well as the resulting hirudin.

The anticoagulant activity present in the salivary glands of the hippopotamus, Hirudo medicinalis, is due to a small polypeptide called hirudin (1). This highly specific and potent thrombin inhibitor has recently been the subject of much research because it represents potentially very interesting therapeutic agents. However, its isolation and purification has been extremely difficult and costly, which has prevented its wider use, and even its clinical examination.

The possibility of producing hirudin by gene cloning, and its expression by recombinant DNA technology, has already been presented by cloning a gene encoding 20 wild-type natural hirudin and expressing it in E. coli (French Patent Application No. 84 04755, March 27, 1984). Although it has been possible to produce a biologically active peptide in E. coli, it is very important to produce hirudin in other microorganisms. In fact, the clinical use of hirudin requires the product: 25 very high purity, and the removal of pyrogenic contaminants can cause problems in purifying hirudin from the coliform bacterial extract.

In addition, hirudin synthesized by E. coli remains intracellular, whereby it has to be purified from a very large amount of E. coli peptide. For these reasons, it would be interesting to express the hirudin gene in yeast, which produces no pyrogenic or toxic substances in humans and is capable of secreting proteins into the culture medium.

The mechanism of action of hirudin as an anticoagulant has only been partially understood. The binding substrate for hirudin is thrombin, a proteolytic enzyme that, upon activation (by activated factor X), of its zymogen-35 form, prothrombin, removes fibrogen from the bloodstream by converting it to fibrin, which is required to form a blood clot. The dissociation constant (0.8 x 10 10) of the thrombin-hirudin-2 104635 (1: 1) complex shows an extremely strong intermolecular association (2). In practice, the non-covalent complex between the two molecules can be considered as undissociated in vivo.

Hirudin is a highly specific thrombin inhibitor with a much higher affinity than the natural substrate, fibrinogen. In addition, other coagulation factors and other plasma components are not required. The specific and very important anti-thrombin activity of hirudin provides the basis for its clinical use as an anticoagulant.

The anticoagulant properties of hirudin have been studied in 10 important ways in animals. The most detailed study (3) describes the activity of hirudin in the prevention of venous thrombosis, vascular occlusion and intravenous growing clots (DIC) in the rat. Hirudin is well tolerated in rats, dogs, rabbits and mice when administered in a highly purified form and by intravenous injection. In mice, the LD50 is greater than 500,000 U / kg body weight 15 (i.e., 60 mg / kg). Another study (4) shows that mice can tolerate up to 1 g / kg and that rabbits can tolerate up to 10 mg / kg both intravenously and subcutaneously. Repeated injections over a period of two weeks do not result in exposure reactions in mice.

In addition, hirudin is rapidly eliminated in experimental animals (half-life of approximately 1 hour) in a still biologically active form by the kidneys (3).

Two other independent studies, one using dogs (5) and the other (6) demonstrating hirudin activity in DIC prophylaxis in rats, are consistent with Markwardt et al. with positive results. These researchers recently published the first in vivo analysis of the effects of natural hirudin on the human hemostatic system (7). The material showed the expected biological effects without showing secondary toxic effects.

It has also been shown that hirudin prevents endotoxin-induced DIC in pigs (8) and thus offers a possible solution to the very serious problem of endotoxin-induced epidemic leading to increased mortality in pigs.

Only one very recent publication (9) describes the intravenous and subcutaneous administration of hirudin to man. Six volunteers have been used to evaluate the pharmacokinetics of a single dose of hirudin (1000 AT-U / kg) and its effect on the hemostatic system. Intravenous hirudin 35 shows a half-life of 50 minutes and 50% of hirudin is found in active form in urine within 24 hours of injection. 3,104,635 prolongation of the clotting time (as measured in vitro for thrombin, thromboplastin and prothrombin) relative to plasma hirudin concentration was observed, indicating that the molecule maintains its biological activity in the subject's circulation. No changes in platelet count in fibrinogen ratio or 5 fibrinogen were observed. Both subcutaneous and intravenous injections of hirudin were well tolerated and did not cause secondary effects. When examining the expression of possible allergic reactions, two skin injections were given to the same subjects every four weeks; no sensitization was observed. In addition, no serum antibody to hirudin was detected.

These studies indicate that hirudin could form a clinically interesting substance as an anti-coagulant. Due to its high specificity, hirudin has no effect on the initial stage of blood clotting. The anti-thrombin effect is dose dependent and the effect of hirudin is rapidly reversible due to its rapid renal elimination. It has been shown that hirudin is much better than heparin for the treatment of DICs (3, 6), as might be expected when DIC status is recognized by antithrombin-III (a cofactor required for heparin function) and elevated coagulation factor 4, which has a very potent anti-heparin effect.

One study has shown the potential for hirudin to be absorbed through human skin (10), although the results are somewhat difficult to interpret.

There are commercial, cell-free creams containing crude leech extract (Hirucreme, Societe Nicholas, France; Exhirud-Blutgel, Plantorgan Werke, SLT), but additional dose testing is still needed:: 25 with highly purified material to ensure that it is an interesting dosage form. Commonly preferred modes of administration are intravenous or intramuscular injections and transdermal administration. Other forms of administration of hirudin have been reported, particularly oral (BSM No. 3,792M).

This product may be used in combination with other compounds for the treatment of psoriasis and other similar dermatological disorders, as disclosed in DOS 2,101,393.

In addition, hirudin can be used as an anti-coagulant in clinical laboratory tests and as a research tool. In this case, the high specificity for a particular stage of blood coagulation may present 4,104,635 significant advantages over commonly used anti-coagulants, which are much less specific.

In addition, hirudin can be very useful as an anti-coagulant in extracorporeal circulation and in dialysis systems where it can represent significant advantages over other anticoagulants, especially if it can be immobilized in active form on the surfaces of the artificial circulation system.

In addition, the binding activity of hirudin to thromblin allows indirect protection of coagulation factors such as Vili during purification.

Finally, the use of labeled hirudin could represent a simple and effective method of measuring thrombin to prothrombin ratios. Specifically, labeled hirudin could be used to visualize the build-ups, since the clotting phenomenon results in the conversion of circulating prothrombin to a troambin at the clotting site, and when the labeled hirudin attaches to the tormbin, this enables visualization.

The invention provides a yeast containing a DNA sequence encoding hirudin, characterized in what is set forth in claim 1. The invention also relates to a process for preparing hirudin with this yeast. The process of the invention is characterized in what is set forth in claim 16.

In summary, the hirudin produced in accordance with the invention presents a number of potential applications: 1) as an anticoagulant in critical thrombotic conditions, prophylactically, and as an inhibitor of the growth of existing thromboses; • f?. ' 2) as an anticoagulant to reduce hematomas and bruising after microsurgery in rooms where live leeches are used; 3) as an anticoagulant in extracorporeal circulatory systems and as an anticoagulant in the coating of synthetic biomaterials; 4) as an anticoagulant in clinical blood samples in laboratory tests; . 30 5) as an anticoagulant in a clinical trial of blood coagulation and as a research tool; '6) as a potential topically active agent in the treatment of skin hemorrhoids, varicose veins and edema; 7) as an active ingredient in the treatment of psoriasis and other skin diseases; 8) Finally, hirudin can be used to fix thrombin in blood storage and in the preparation of blood derivatives (platelets, factor Vili and IC).

As an active ingredient, hirudin can be used in therapeutic compounds at concentrations of 100-50,000 U / kg / day of antithrombin.

With the water solubility of hirudin, it is easy to produce injectable or otherwise administrable pharmaceutical compounds using pharmaceutically acceptable carriers and media.

Finally, it is possible to use labeled hirudin, either radiolabeled or by any other known enzymatic or fluorescent labeling method, for in vitro dosing determination or in vivo monitoring, particularly to visualize clot formation.

A whole animal derived hirudin preparation has been used to determine the amino acid sequence of a protein (11, 12). The following experiments contain a cloned gene that is expressed as messenger RNA at the end of the leeches during fasting. This gene carries information from a protein (hirudin variant 2 or HV-2) that has a sequence substantially different from that found in the body of 20 animals (called the protein variant HV-1). HV-1 and HV-2 differ by 9 amino acid residues, and differences between the NH2 terminals (val-val or Ile-thr) are likely to be explained in the literature with respect to the NH2-terminus of hirudin (13).

Figure 1 shows the DNA sequence of the recombinant plasmid pTH717, which contains a copy of the cDNA corresponding to the HV-2 mRNA, 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-T.

It is necessary to note that the cDNA sequence is unlikely to be complete and that it may also be preceded by a signal sequence at the beginning of a working protein.

The expression of HV-2 cDNA in microorganisms indicates that the corresponding v protein has antithrombin activity.

Although the following experiments were carried out with variant HV-2, "hirudin" and "hirudin-encoding gene" hereinafter refer to both variants, i. HV-1 or HV-2, as well as any other variants, and sequences corresponding thereto, unless otherwise stated.

e 104635

One aspect of the present invention is the production of hirudin in yeast.

Yeast is a single-celled eukaryotic organism. The yeast species Saccharomyces comprises strains that have been intensively studied biochemically and genetically in laboratories; it also includes strains used in the food industry (bread, alcoholic beverages, etc.) which are therefore produced in very large quantities.

Saccharomyces cerevisiae cells are easily manipulated genetically, either by classical methods or by genetic engineering, or more preferably by combining these two techniques, and when the species has a long industrial background, it becomes a preferred host for the production of foreign polypeptides.

Therefore, the present invention relates in particular to a functional DNA fragment which permits production of hirudin in yeast, comprising at least: a gene for hirudin or one of its variants (hereafter the H gene); a DNA sequence (Str) containing the signal sequence, which allows transcription of the H gene in yeast.

This functional fragment, when integrated into a plasmid or chromosome of a yeast, preferably a Saccharomyces species, could allow, after transformation of the aforementioned yeast, expression of hirudin, either as an active form or as an inactive hirudin-activating regenerative precursor.

Saccharomyces cerevisiae is interesting because this yeast is able to secrete some proteins in its culture environment; the mechanism responsible for this phenomenon is constantly being studied and it has been shown that it is possible: to make the yeast, after the necessary manipulation, secrete human hormones properly processed and fully equivalent to those found in human serum (14, 15).

The present invention takes advantage of this property to achieve the secretion of hirudin because of the many advantages that this brings.

30 First, yeast secretes little protein, which is advantageous if v can control the secretion of foreign protein to a high level to produce a product that represents a high proportion of the total · - “total protein secreted, thus facilitating purification of the protein under study.

There are several yeast secreted proteins or polypeptides. In all known cases, proteins are synthesized at length 104635 as a precursor form whose NH2 terminal sequence is crucial for the selection of the metabolic pathway leading to excretion.

Synthesis of hybrid proteins containing a precursor NH2 terminal and subsequent foreign protein sequence in yeast can, in some cases, lead to the secretion of this foreign protein. The fact that the foreign protein is synthesized in the precursor form, and thus generally inactive, allows the cell to be protected against the potential toxic effects of the molecule being studied, liberating the active protein only in vesicles formed in the Golgi device that isolate the protein from the cytoplasm.

The use of metabolic pathways leading to secretion to produce foreign protein in yeast thus has several advantages: 1) it allows the recovery of a relatively pure product from a culture supernatant; 2) it allows the cell to be protected against potential toxic effects of the functional protein.

3) In addition, in some cases, the secreted proteins may be modified (glycosylated, sulfated, etc.).

Therefore, the expression fragments used in the invention most preferably have the following structure: 20 - S * - L * - Sd - H gene - wherein Lex encodes a "leader" sequence for secretion of a protein corresponding to the H gene; : 25 Sd is the DNA sequence encoded by the cleavage site; in addition, the S1H gene region may be repeated several times.

For example, alpha-pheromone has been selected for the secretion system, i.e., in the above sequence, the L1 sequence represents the yeast alpha-sex pheromone, but other systems could be used (for example, 30 Killer protein systems) (14).

The yeast alpha sex pheromone is a 13 amino acid peptide (boxed in Figure 2) that is secreted into the culture medium in the S. cerevisiae yeast sexual Mata. The alpha factor stops cells of the corresponding sex species (Mata) to enter G1 and induces the biochemical and morphological changes required for cell type association. Kurjan and Herskowitz (17) have cloned the alpha factor structural gene and have deduced from the gene sequence that this 13 β 104635 amino acid factor is synthesized as a 165 amino acid pre-pro-protein precursor (Fig. 2). The precursor contains a hydrophobic amino terminal sequence of 22 residues (underlined by a dashed line) followed by a 61 amino acid sequence containing 3 glycosylation sites followed by 5 alpha factor 4 copies. The "Spacer" sequences separate the copies and release the functional protein from the precursor by the following enzymatic action: 1) a cathepsin B-like endopeptidase that cleaves the COOH-terminated dipeptides Lys-Arg (indicated by bold arrow); an exopeptidase that cleaves 10 basic residues at the COOH end of the truncated peptides; 3) a dipeptidylaminopeptidase (A) which removes residues Glu-Ala and

Asp-Ala.

The precursor nucleotide sequence further contains 4 HindIII restriction sites, designated Hilla in the figure.

Several fusions between the α-pheromone gene and the functional hirudin sequence have been performed. Mata-type yeast cells are capable of expressing these fusion genes. The corresponding hybrid proteins can thus be processed by the signals they contain which contain the pre-pro sequence of the α-pheromone precursor. Thus, it is expected that polypeptides having the hirudin sequence can be recovered from the culture supernatant.

In one construct, the Sd sequence contains at its 3 'end an ATG codon upstream of the H gene; the fusion protein thus contains methionine, which functions above the first amino acid of hirudin. After cleavage with cyanogen bromide, this polypeptide forms a hirudin molecule that is • converted to its active form after the renaturation step.

Other constructs use cleavage signals commonly used in the production of α-pheromone to produce polypeptides with antithrombin activity in the culture supernatant. This is accomplished when the Sd sequence contains at its 3 'end two codons encoding Lys-Arg, i.e.. AAA or AAG and 30 AGA or AGG; the polypeptide is cleaved by endopeptidase, which cleaves the Lys-Arg dipeptides at the COOH terminus to release hirudin.

In particular, the present invention uses constructs in which the sequence preceding the hirudin gene encodes the following amino acid sequences: 1) Lys Arg Glu Ala Glu Ala Trp Leu Gin Val Asp Gly Ser Met Hirudin

... I

9 104635 2) Lys Arg Glu Ala Glu Ala Hirudin ..., 3) Lys Arg Glu Ala Glu Ala Lys Arg Hirudin ..., 4) Lys Arg Glu Ala Glu Ser Leu Asp Tyr Lys Arg Hirudin ... or 5) Lys Arg Hirudin ...

Of course, it is possible to contemplate the use of other sequences selectively cleaved by the enzyme at the amino acid level, provided that the cleavage site does not occur in the hirudin itself.

Finally, the expression blocks may contain, after the H gene, a yeast termination sequence, for example, a PGK gene termination sequence.

In general, the expression fragments used in the present invention may be integrated in yeast, particularly in the Saccharomyces species, either in an autonomously replicating plasmid or in a yeast chromosome.

When the plasmid is autonomous, it contains replication-enabling moieties, i. the origin of replication, for example, from plasmid 2μ. Additionally, the plasmid may contain selectable elements, such as the URA3 or LEU2 genes, which ensure the ura3 or leu2 complementation of the yeast strains. These plasmids may also, when the plasmid is so-called. "shuttle" plasmid, contains portions that allow their replication in bacteria, such as the origin of replication of pBR322, a marker gene such as Ampr and / or other elements known to those skilled in the art.

The present invention also relates to yeast strains transformed with said expression fragments, wherein the fragment is either in a plasmid or integrated into chromosomes. These yeasts should be mentioned in particular

Yeasts of the species Saccharomyces, in particular S. cerevisiae.

: 25 When the promoter is an α-pheromone gene, the yeast of the sex type Mata is preferably used. For example, a strain that is genotyped with ura3 or Ieu2 or another plasmid complementable strain is used to maintain the plasmid in yeast by appropriate selection pressure.

Although it is possible to produce hirudin by fermentation of the transformed strains mentioned above in modified medium by accumulating hirudin into cells, it is more advantageous, as shown in the above discussion, to expose hirudin to the environment, either as a functional form or as a precursor form. vitro.

This maturation can be performed in several steps. First, it may be necessary to cleave certain elements derived from the translation of the Lex sequence, which is carried out at a level similar to that of the Sc1 sequence. As disclosed above <n 10463510, a functional hirudin may be preceded by methionine, which is selectively cleaved by cyanogen bromide. This method is useful because the sequence encoding hirudin does not contain methionine.

The N-terminus may also have a Lys-Arg dipeptide which is cleaved from the 5 COOH-terminals by a specific endopeptidase; this enzyme acts during the secretion process, thereby providing the working protein directly to the environment.

But, in certain cases, it may be necessary to carry out the enzymatic digestion after excretion by adding a specific enzyme.

In some cases, especially after treatment with cyanogen bromide, it may be necessary to renaturate the protein for the re-formation of disulfide bridges. The peptide is then denatured, for example with guanidine hydrochloride, and renatured in the presence of reduced glutathione and oxidized.

Other features and advantages of the present invention will be apparent from the following examples.

15 Attached Patterns:

Figure 1 shows the nucleotide sequence of the hirudin cDNA fragment cloned into plasmid pTG 717; Figure 2 shows the nucleotide sequence of a precursor of the? Figure 3 illustrates the construction of plasmid pTG834; Figure 4 illustrates the construction of plasmid pTG880; Figure 5 illustrates the construction of plasmid pTG882; Figure 6 illustrates the construction of the phage MI3TG882; Figure 7 illustrates the construction of plasmid pTG874; . Figure 8 illustrates the construction of plasmid pTG876; Figure 9 illustrates the construction of plasmid pTG881; Figure 10 illustrates the construction of plasmid pTG886; Figure 11 illustrates the construction of plasmid PTG879; Figure 12 shows an acrylic amide electrophoresis gel containing PM> 1000 proteins isolated from a culture medium of yeast transformed with plasmids pTG886 and pTG897; Figure 13 shows an acrylamide electrophoresis gel containing protein PM> 1000 isolated from a culture medium of yeast transformed with pTG886 and pTG897; Figure 14 illustrates the construction of plasmid pTG1805; 104635 11 Figure 15 shows an acrylamide electrophoresis gel containing proteins PM> 1000 isolated from a culture medium of yeast transformed with pTG847 and pTG1805; Figure 16 is a sketch comparing the amino acid sequences below the first cleavage site 5 (Lys-Arg) ("en Aval") in different constructs.

The amino acid sequences and nucleotide sequences are not linked to, but are still part of, the present disclosure.

Example 1 Construction of plasmid PTG822

Plasmid pTG717 was digested with PstI, then with Hinfl and Ahall1. followed by isolation of the hirudin HV-2 cDNA fragment from the gel. The enzyme Hinfl cleaves the hirudin-HV-2 sequence downstream of the first codon. The enzyme Ahall1 cuts about 30 base pairs behind the 15 stop codons (3 ') of the hirudin sequence. The Hinfl-Ahall1 fragment thus obtained was isolated on an agarose gel and eluted from the gel.

The coding sequence for the functional protein was inserted into vector pTG880. The vector pTG880 is a derivative of vector pTG838 (Figure 3). Plasmid pTG838 is identical to pT833 except for the Ballllk site, which is located near the 20 transcription termination site of PGK. This site is inactivated by Klenow polymerase to give pTG838.

Plasmid pTG833 is a shuttle plasmid for yeast and E. coli. This plasmid is constructed to express foreign genes in yeast. The basic units of this vector (Figure 3) are the following: URA3 gene as a yeast selection marker, 25 yeast 2μ plasmid replication origin, E. coli ampicillin resistance gene and pBR322 plasmid replication origin (these latter units allow production of plasmid g), A 5 'flanking region encoding this gene up to the SalI site, the Sall-Pvull phrase of pBR322, and the termination site of the PGK gene (20). This plasmid was previously described in FR Patent Application No. 84 07125 filed May 9, 1984 in the name of the Applicant.

Plasmid pTG880 is constructed from pTG838 (Fig. 4) by inserting a short polylinker region (derived from bacteriophage M13) into the plasmid pTG838 digested with EcoRI and BglII, allowing immediate sequencing of the following 35 cloning sites, BGIII, PstI, HindIII, after the 5 'flanking region of the yeast PGK gene. The DNA of plasmid 12 104635 pTG880 was digested with BglII and SmaI and the large fragment generated by digestion was isolated and eluted from the gel. The Hinfl / Ahall1 fragment of plasmid pTG717 containing the major part of the sequence encoding hirudin HV-2 was mixed with the digested pTG880 together with 3 synthetic 5 oligonucleotides (Figure 5) designed to reconstruct the NH2-B of the hirudin sequence and hinudin-containing fragment of hirudin. This mixture was subjected to ligation treatment and then used to transform E. coli cells. Transformants were recovered from plasmid pTG882.

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

Example 2 Construction of PTG886 and dTG897

First, the BglII-EcoRI fragment (230 bp) of pTG882 containing the hirudin sequence was inserted into the bacteriophage M13mp8 (Figure 6) between BamHI and EcoRI. There was thus obtained a phage M13TG882 from which an EcoRIHindIII fragment (about 245 bp) could be isolated.

Plasmid pTG881 (10 kb) is an E. coli / yeast shuttle plasmid that replicates autonomously in both E. coli and Saccharomyces cerevisiae, uvarum and carlbergens strains.

Plasmid transfer to E. coli confers resistance to ampicillin (and other β-lactam antibiotics). In addition, this plasmid contains the 30 yeast LEU2 and URA3 genes. which are expressed in E. coli and • l '. Saccharomyces strains. Thus, the presence of the plasmid in E. coli or f

In Saccharomyces, it allows complementation of B-isopropyl malate dehydrogenase or OMP-decarboxylase-free strains.

Plasmid pTG881 was constructed as follows: 104635 13 The starting plasmid was pTG848 (identical to pTG849 disclosed in FR Patent 8315716 except for the ura3 gene, which is composed of the following DNA fragments (Figure 7): 1) Approximately 3, 3 kb EcoRI-HindIII fragment from pJDB207 (18).

The HindIII site corresponds to coordinate 105 in the 2µ-ρΐ38ΓΤ ^ ίη B form, the EcoRI site corresponds to coordinate 2243.

2) HindIII fragment of the URA3 gene (19).

3) pBR322 large EcoRI (coordinate 0) -Sall (coordinate 650) fragment. A 510 bp EcoRI-HINDIII fragment (whose ends were first blunted by Klenow in the presence of 4 nucleotides) corresponding to the remainder of the PGK gene (20) was inserted into the Pvull site of this fragment. The blunted EcoRI end of the PGK gene regenerates the EcoRI site when inserted into the 15 Pvull ends of pBR322.

4) HindIII-SalI fragment of PGK gene (2.15 kb) (20).

Plasmid pTG848 was digested with Hindu and the ends of the fragments thus formed truncated by Klenow treatment in the presence of 4 deoxyribonucleotides. The fragments were ligated and the ligation mixture treated with Hindu prior to 20 transformations, thereby deleting all plasmids retaining one (or two) HindIII sites. E. coli strain BJ5183 (pyrF) was transformed and transformants were selected for their ampicillin resistance and pyr 'property.

Thus, plasmid pTG874 (Figure 7) was obtained in which both HindIII sites are inactivated, the orientation of the URA3 gene gives transcription in the same orientation as the phosphoglycerate kinase gene (PGK).

Plasmid pTG874 was digested with SmaI and SalI and the 8.6 kb fragment was isolated from an agarose gel. Plasmid pTG864 containing the Eco RI-Sal I fragment (about 1.4 kb) of the MF 1 gene cloned at the same sites in pBR322 was digested with Eco RI. Thus, the ends of the linearized plasmid were truncated by Klenow treatment in the presence of 4 nucleotides. This was followed by SalI digestion and the EcoRI (blunt) SalI fragment corresponding to the MF1 gene was isolated. The latter fragment was ligated into pTG874 i

Smal-SalI fragment (8.6 kb) to give plasmid pTG876 (Figure 8).

To inactivate the MFα1 promoter sequence, proximal I

The BglII site was subjected to partial digestion of plasmid pTG876 with BglII followed by Klenow treatment in the presence of 4 deoxyribonucleotides. The resulting new 10463514 plasmid, pTG881 (Fig. 9), permits insertion of foreign coding sequences between the first HindIII site of the MFα1 gene and the BglII site at the end of the PGK gene.

When the addition of foreign encoding DNA to HindIII site 5 allows translation in the same reading step, the resulting hybrid protein contains pre-pro portions of the alpha-pheromone.

Cloning of the HindIII-EcoRI fragment containing the hirudin gene into pTG881 gives plasmid pTG886 (Figure 10). Once the cloning has been completed, the BglII site is located underneath the fragment, which allows for the removal of the 10 fragments in the Hinfl-BglII form, whereby the Hinfl site is the same as used above. With BglII present only once in pTG881, it is very easy to reconstruct the 5 'end of the hirudin coding sequence using 3 oligonucleotides that reconstruct the hirudin 5-terminus and allow the correct reading of the α-pheromone pre-pro sequence and subsequent functional hirudin.

This new plasmid is called pTG897 (Figure 11).

Example 3

Expression of hirudin in yeast

Plasmid pTG897 DNA was used to transform the yeast TGY1sp4 20 (Mata, ura3 -251-373-328, his3-11-15) into the ura + form using the above procedure (21).

The transformed colony was picked and inoculated into 10 ml of minimal medium supplemented with casino acids (0.5%). The culture was continued for 20 hours, after which the cells were centrifuged, the supernatant dialyzed with distilled water. 25 and concentrated by evaporation (centrifugation in vacuo).

In parallel, the TGY1sp4 culture transformed with plasmid pTG886 and the culture transformed with plasmid without hirudin (TGY1sp4 pTG856) were similarly treated. The dry pellets were taken up in 50 µl of water and 20 μΙ were boiled in the presence of 2.8% SDS and 100 mM mercaptoethanol and placed on an acrylamide-SDS gel (15% acrylamide; 0.1% m / v SDS) (22). After attachment and Coomassieblue staining, polypeptides accumulated in the supernatant of the TGY1sp4 / pTG886 and TGY1sp4 / pTG897 cultures, which are absent from the control culture supernatant, could be detected (Figure 12). In addition, additional sets of peptides are labeled with strong 35S-cysteine, as might be expected from a hirudin peptide because this molecule is highly cysteine-rich (Figure 10).

is 104635

The electrophoresis shown in Figures 12 and 13 was performed as follows:

For Figure 12, extracts were prepared as follows: 10 ml of minimal medium (Yeast Nitrogen Base Difco without amino acids (6.7 g / l), glucose (10 g / l) supplemented with 0.5% casino acids, inoculated with different strains and cultured 20 hours (stationary phase) Cells were centrifuged, supernatant was dialyzed against water (minimum retention: PM 1000) and dried by vacuum centrifugation. Samples were then taken in 50 μΙ gel buffer, from which 20 μΙ was treated as above and transferred to acrylamide SDS (15%). acrylamide, 0.1% SDS) (22).

10 The heels used were:

Lane 2: TGY1sp4 transformed with a plasmid lacking the hirudin sequence (control);

Lane 3: TGY1sp4 transformed with pTG886;

Lane 4: TGY1sp4 transformed with pTG897; Lane 1: Reference marks were added to this lane (Pharmacian LMW Krt; top to bottom; 94,000, 67,000, 4300, 30,000, 20100, 14,000). , ..

The bands were detected by staining with Coomassie-blue R-250.

For Figure 13, samples were prepared as follows: 100 ml of 20 minimal medium + histidine 40 mg / ml were inoculated with different strains and cultured overnight. When the cell density reached about 5 x 10® (putative exponential phase), 40 μΙ of 35-cysteine (9.8 mCl / ml; 1015 Ci / mmol) was added to each culture. After 10 minutes, the cells were centrifuged, harvested in 10 ml complete medium (30 ° C) and incubated at 30 ° C with shaking. After 3 hours, 10 ml of supernatant was dialyzed against water and concentrated to 0.5 ml as shown in Figure 12. Approximately 35,000 cpm (40 μΙ) was transferred to an acrylamide SDS gel (15% acrylamide, 0.1% SDS). Protein bands were visualized by fluorography.

The strains used were:

Lane 2: TGY1sp4 transformed with no plasmid. , 30 hirudin sequences;

Lane 3: TGY1sp4 transformed with pTG886;

Lane 4: TGY1sp4 transformed with pTG897;

Lane 1: Markers with reported PM (x103) were added to this lane.

Similarly, when supernatants containing polypeptides were tested for antithrombin activity, no activity was observed.

104635 For the polypeptide secreted by the 16 TGY1sp4 / pTG886 strain, the pheromone precursor, cleaved by a standard method, was expected to have a hirudin molecule with 8 additional amino acids at its NH2 terminus. Thus, it is not surprising that the polypeptide secreted by TGY1sp4 / pTG886 cells is not active.

On the other hand, for the polypeptide secreted by TGY1sp4 / pTG897, it is expected that the polypeptide will be identical to the native protein and thus active. Several hypotheses may explain the lack of activity: 1) Protein naturation is incomplete, there may still be 10 Glu-Ala residues at the NH2 terminus as shown for EGF (16).

2) The protein is inactive because it does not have the correct conformation or the culture supernatant contains proteins or other molecules of the size PM 1000 that inhibit the activity.

3) The protein is incomplete because it has undergone intracellular and extracellular proteolysis.

Example 4

Activation by Svanoaen Bromide Cleavage

If the lack of activity is due to the additional NH2-terminal amino acids, it should be possible to obtain the activity by subjecting the peptide secreted by TGY1sp4 / pTG886 to cyanogen bromide treatment. This reagent is specific for methionine residues and the fusion protein encoded by pTG886 does not contain any methionine. This reaction was performed as follows: Yeast cells containing either pTG886 plasmid or control plasmid containing no hirudin insert were cultured in 10 ml medium for 24 hours. By this time, culture 25 had reached a density of 7-10 x 10 7 cells / ml. The supernatant was separated from the cells, dialyzed against distilled water and lyophilized. The dry powder was dissolved in 1 mL of 70% formic acid and a portion was used to determine total protein (using Coomassie-blue staining, reagents purchased from Biorad); the remainder of the sample was treated with 1 ml of fresh 30 cyanogen bromide solution (30 mg / ml) in 70% formic acid.

t

Oxygen was removed by nitrogen purge and the tubes incubated in the dark for 4 hours at room temperature. All necessary precautions were taken in the presence of cyanogen bromide and in the fume cupboard. The cyanogen bromide cleavage reaction was stopped by the addition of 10 volumes of 35 distilled water, followed by lyophilization.

17 104635

The digested peptide was redissolved in 10 ml of distilled water and lyophilized twice more. Finally, the peptides were dissolved in a small amount of distilled water and a portion was used to determine antithrombin activity. The rest of the sample was lyophilized and subjected to the renaturation steps shown below.

Because hirudin activity is dependent on the presence of disulfide bonds in the molecule (1), it seems likely that the cyano-bromide-cleaved peptide will need to be properly renatured for biological activity to occur. Therefore, the digested peptides were subjected to denaturation with 5 M GuHCl, followed by renaturation using 10 methods known to those skilled in the art.

In summary, the lyophilized peptides were dissolved in 400 µl of 5 M guanidine hydrochloride (GuHCl) in 250 mM TrisHCl, pH 8.9; then, the solution was brought to 2 mM with reduced glutathione and 0.2 mM with oxidized glutathione in a total volume of 2.0 mL (final concentration 1.0 M GuHCl and 50 mM Tris).

After incubation for 16 hours at 23 ° C in the dark, the samples were dialyzed 24 times three times with 2 L of 50 mM Tris-HCl, pH 7.5, 50 mM NaCl at 23 ° C, and finally the dialysis solution was clarified by centrifugation.

The antithrombin activity of the supernatants was measured. The result of this experiment, shown in Table I, clearly shows an increase in anti-thrombin activity in the supernatant of cells infected with Gli with plasmid pTG886, while no activity in control plasmids.

TABLE I

:, 25 Antithrombin activity of supernatants of yeast cultures

specific

Supernatural Activity Activity U / mg

Plasmid Activity U / ml_Of Protein pTG886 a) After Cleavage, Before Renaturation <0.3

• i I

* · 'B) after cleavage and renaturation 2.46 102.5 controls after a) cleavage, before reaturation <0.3 b) after cleavage and renaturation <0.15 <5.6 18 104635

In conclusion, the yeast cells having the recombinant plasmid may secrete a peptide having the biological activity of hirudin following the cleavage reaction and renaturation. This indicates that the presence of additional amino acids at the NH 2 terminal is sufficient to explain the lack of activity of the polypeptides in cultures for TGY1sp4 / pTG886 and possibly also for TGY1sp4 / pTG897 cultures (GluAla repetition).

Example 5

Insertion of a new cleavage site just before the first amino acid in the hirudin-HV2 coding sequence - plasmid oTG1805 In construct pTG897, there is a risk that the secreted peptide still has Glu-Ala tails at the NH2 end, which would explain the lack of antithrombin activity here. If this hypothesis is true, it must be possible to activate the peptide directly in the supernatant by creating a cleavage site between the Glu-Ala residues and the first amino acid 15 (isoleucine) of hirudinHV-2. This is accomplished by the addition of the dipeptide LysArg coding sequence, which is an endopeptidase recognition site in the ferromin precursor maturation (Figure 2). The construct was obtained in exactly the same manner as shown for plasmid pTG897, except for 2 synthetic oligonucleotides (Figure 14). The resulting plasmid was called pTG1805. The plasmid was used to transform the TGY1sp4 strain into groove +. The material secreted into the supernatant was analyzed on an electrophoresis gel as above and compared with the material obtained with TGY1sp4 / pTG897 (Figure 15).

The strains used were:

Lane 1: markers identical to those used in Figure 12; Lane 2: TGY1sp4 transformed with pTG897;

Lane 3: TGY1 sp4 transformed with pTG1805;

Lane 4: Control that does not produce hirudin.

Polypeptides specific for strain TGY1sp4 / pTG1805 move more slowly than those specific for strain TGY1sp4 / pTG897. This result indicates that the corresponding endopeptidase does not efficiently use the new cleavage site. However, the level of hirudin in the supernatant as measured by biological activity reveals that a small portion of the material is in active form, in contrast to TGY1 in sp4 / pTG987, which is clearly inactive (Table II).

19 104635

TABLE II

Antithrombin activity of yeast supernatants (10 ml) Selective activity Total activity

Plasmid_U / mg_U (10 ml) pTG856 not measurable pTG897 not measurable pTG1805 21 2.0

Example 6 A new construct with a new more efficient cleavage site. which enables the release of a functional hirudin

In the previous construct (pTG897), no low hirudin activity in the supernatant was obtained, probably because another cleavage site releasing active hirudin is identified at low 10 potency. Therefore, a new construct has been made to make this additional cleavage site more endopeptidase sensitive by inserting a sequence above it, a three amino acid, Ser Leu Asp, which naturally occurs above the first LysArg dipeptide (Figures 2 and 16).

The method used is the same as above except for the 15 oligonucleotides which have the following sequences: 26-mer 15-mer 5'-AGCTTCTTTGGATAAAAGAATTACGT ^ TACAGACTGCACAG * 20 .AGAAACCTATTTTCTTAATGCATATGTCTGACGTGTCTta is shown in Fig.

· *; The corresponding plasmid is called pTG1818, and differs from pTG1805 only by inserting the corresponding nucleotides of the Ser-Leu-Asp codons: 5-TTG GAT AAA.

The activity measured in the supernatant of TGY1sp4 / pTG1818 cultures under the standard conditions previously described is about 200 units per 10 ml culture, which is about 100 times higher than in the previous 104635 Example. It should be noted that the assay can be performed on 10 µl of non-concentrated culture medium.

Example 7

Construction that results in precursor synthesis lacking the Glu-Ala-Glu-Ala sequence

In the previous two examples, it has been shown that the addition of a new Lys Arq site above the beginning of the sequence of the just working hirudin allows the release of active material into the supernatant. However, in these examples, heavier inactive contamination on the culture medium can be obtained by forming hirudin chains with extended NH2 terminals if precursor cleavage occurs at the first site of LysArg (the site of distal function). This is particularly evident for TGY1sp4 / pTG1805 cultures where the inactive contaminant predominates. However, this is equally possible with TGY1sp4 / pTG1818-15 cultures where the inactive contaminant is not predominant but where it may occur, as others have suggested in the case of EGF (15). In order to avoid this loss in the yield of synthesis of the inactive material, it was decided to carry out a novel construct which gives a precursor that is not different from the synthesis of TGY1sp4 / pTG897 cells except that the Glu-Ala-Glu-Ala sequence is absent in Lys-Arg: n cleavage site and the first amino acid of the functional hirudin sequence.

The following strains have been deposited in the INSTITUT PASTEUR collection "Collection Nationale de Cultures de Microorganismes (CNCM)", 28 Rue du Docteur-Roux, PARIS 15'eme, April 30,1985: •. TGY1sp4 / pTG1818: Saccharomyces cerevisiae strain TGY1sp4 (Mata ura3-251-373-328-his 3-11-15) transformed into ura + strain with plasmid pTG1818; deposit # 1441.

TGY1sp4 / pTG886: Saccharomyces cerevisiae strain TGY1sp4 Mata ura3-251-373-328-his 3-11-15) transformed into ura + strain by plasmid pTG886; deposit # 1442.

*

References 1. BAGDY D., BARABAS E., G RAF L, PETERSEN T.E. and MAGNUSSON S. (1976) Methods in Enzymology part B, vol. 45, p. 669-678.

35 2. MARKWARDT F. (1970) Methods in Enzymology, ed. Perlman G.E. and Lorand L, Academic Press., vol. 19, p. 924-932.

104635 21 3. MARKWARDT F., HAUPTMANN J., NOWAK G., KLESSEN Ch. and WALSMANN P. (1982) Thromb. Hemostasis (Stuttgart) 47,226-229.

4. WALSMANN P. and MARKWARDT F. (1981) Die Pharmazie 10, 653-660.

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(1980) Thrombosis Research 19, 351-358.

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10. SUTOR A.H., KNOP S. and ADLER D. (1981) Kontralle Antithrombotica, 23rd Symp. Blutgerinnung, Hamburg, p. 117-123.

15 11. PETERSEN T.E., ROBERTS H.R., SOTTRUP-JENSEN L, MAGNUSSON

S. and BADGY D. (1976) Protides Biol. Fluids, Proc. Colloq. vol. 23, p.

145-149.

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20 Res. 30, 459-467.

14. BUSSEY H., SAVILLE D., GREENE D., et al. (1983) Mol. Cell. Biol. 3, 1362-1370.

15. BRAKE A.J., MERRYWEATHER J.P., COIT D.G., HEBERLEIN U.A., MARIARZ F.R., MULLENBACH G.T., URDEA M.S., VALENZUELA P. ia •. 25 BARR P.J. (1984) Proc. Natl. Acad. Sci. USA 81, 4642-4646.

16. BITTER G.A., CHEN K.K., BANKS A.R.ja LAI P.H. (1984) Proc. Natl.

Acad. Sci. USA 81, 5330-5334 17. KURJAN J. and HERSKOWITZ I. (1982) Cell 2, 933-943.

18. BEGGS J. (1981) Genetic Engineering 2,175-203.

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Res. 10.7791-7808. ______________ - 21. ITO H., FUKUDA Y., MURATA K. and KIMURA A. (1983) J. Bacteriol. 153, 35163-168.

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Claims (17)

1. A yeast transformed with a functional DNA fragment integrated into a plasmid containing the origin of replication in the yeast or integrated into the chromosome of said yeast, characterized in that the fragment contains at least: - a hirudin-encoding DNA sequence (gene H), - at least one a cleavage site (Sd) located immediately upstream of the gene H, - a leader sequence (L ^ containing the necessary elements for secretion of the gene product), - a DNA sequence (S *) containing the signals that ensure transcription of the H gene by yeast. Transformed yeast according to claim 1, characterized in that said functional fragment contains at least the following sequences, 5'-15-to-3: - a sequence (S *) containing the signals which confirm the transcription of the H gene by the yeast, - a leader sequence (L1) containing the necessary elements for secretion of the gene product, 20. The DNA sequence encoding HVr or HV2-hirudin encoded by the amino acid sequence shown in lines (c) and (b) of Figure 1, respectively, and preceded by the cleavage site Sd. Yeast according to claim 1 or 2, characterized in that the sequence Lw is the yeast alpha-sex pheromone sequence shown in Figure 2. Yeast according to any one of claims 1 to 3, characterized in that the leader sequence contains the yeast alpha-sex pheromone pre-fragment shown by dashed lines in Figure 2. Yeast according to any one of claims 1 to 4, characterized in that the fragment contains the yeast alpha-sex pheromone pre-pro sequence 104635 at the 5 'end of the H gene which is shown in Figure 2 and which encodes amino acids 1-83. Yeast according to any one of claims 1 to 5, characterized in that the H gene is followed by a yeast termination sequence. Yeast according to claim 6, characterized in that the yeast termination sequence is a PGK gene termination sequence. Yeast according to any one of claims 1 to 7, characterized in that the cleavage site is a dipeptide Lys-Arg having the DNA sequence AAA or AAG in combination with AGA or AGG. Yeast according to any one of claims 1 to 7, characterized in that the functional fragment is integrated into a plasmid containing at least one origin of replication in yeast. Yeast according to claim 9, characterized in that the origin of replication is the origin of replication of the 2p plasmid. Yeast according to claim 9 or 10, characterized in that the plasmid further contains a selection property. Yeast according to claim 11, characterized in that the selection property is due to the URA3 gene. Yeast according to any one of claims 1 to 12, characterized in that it belongs to the genus Saccharomyces. Yeast according to claim 13, characterized in that it represents the genus Matalfa. Yeast according to any one of claims 12 to 14, characterized in that it is a strain of S. cerevisiae. A process for the preparation of hirudin, characterized in that the yeast according to any one of claims 1 to 15 is fermented in a culture medium and the hirudin formed is recovered from the culture medium. The method according to claim 16, characterized in that 104635 a) cultivates in culture medium a strain of yeast containing a functional DNA fragment in a plasmid or integrated into the chromosome of said yeast, which fragment comprises at least the sequence: 5 -S * -Lex-Sd-H gene ter where - S * is a DNA sequence containing signals that confirm the transcription of the H gene by yeast, 10. l_ex is a leader sequence that permits secretion of mature hirudin or secretion of hi-rudine as a hirudin precursor that matures in vitro, - Sd is a DNA signal encoding a cleavage site selected from ATG and Lys-Arg coding sequences, b) recovering the resulting hirudin from the culture medium in mature form or hirudin , which matures in vitro. 25 104635