DK174837B1 - Process for producing hirudin as well as transformed yeast which can be used in the process - 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

in DK 174837 B1

The present invention relates to yeasts transformed with vectors for expression of a DNA sequence encoding hirudin or hirudin analogs, and to a method of producing hirudin which is characterized by the characterizing part of claim 16.

5

The anticoagulant effect found in the salivary glands of medical gills, Hirudo medicinalis, is derived from a small polypeptide called hirudin (1). This very specific and very effective inhibitor of thrombin has been studied extensively in recent times as it potentially represents a very interesting therapeutic agent. However, the considerable difficulties and costs associated with isolating and purifying the inhibitor have prevented it from being used more widely and can be investigated at the clinical level.

The ability to produce hirudin by cloning genes and expression of the genes by recombinant DNA technology has been demonstrated by cloning of a natural Igle gene encoding hirudin and by expression of the gene in the E. coli microorganism (French patent application no. .

84 04755 in the name of Transgéne S.A., filed March 27, 1984). Although it has been possible to prepare a peptide with biological activity in E. coli, it is very important to prepare hirudin in other types of microorganisms. Indeed, clinical use of hirudin requires a very high degree of purity of the product, and elimination of pyrogenic contaminants could cause problems in purifying hirudin from extracts of the colibacteria.

In addition, hirudin synthesized by E. coli remains intracellular and thus must be purified from a very large number of E. coli peptides. It is therefore interesting to allow the hirudin gene to be expressed in yeasts which do not form substances which are pyrogenic or toxic to humans and which are capable of secreting proteins in the culture medium.

Hirudin's mechanism of action as an anticoagulant has only recently been recognized. The substrate for hirudin fixation is thrombin, which is a proteolytic enzyme that, upon activation (with the activated factor X) from its zymogenic form, prothrombin, cleaves the fibrin gene in the circuit and converts it into fibrin which is required for formation of clot.

The dissociation constant of complex thrombin / hirudin 1: 1 (0.8 x 10-10) suggests a very strong bond between these molecules (2). In practice, the non-covalent complex between these two molecules can be considered as non-dissociable in vivo.

35

Hirudin is a very specific inhibitor of thrombin with a much greater affinity than the natural substrate, fibrinogen. Furthermore, it is not necessary to have other coagulation factors or other plasma components. Hirudin's specific and very large antithromic bin activity explains its clinical utility as anticoagulants.

40

Hirudin has been studied very extensively in animals for its anticoagulant properties. The most detailed study (3) describes hirudin's activity in preventing vein thrombosis, vascular occlusions and disseminated intravascular coagulations (DIC) in rats. Hirudin is well tolerated by rats, dogs, rabbits and mice when in highly purified form and injected intravenously. LD50 in mice is over 500,000 U / kg body weight (ie 60 mg / kg). Another study (4) shows that mice can tolerate doses up to 1 g / kg and that rabbits can tolerate up to 10 mg / kg both intravenously and subcutaneously. In mice, repeated injections over a 2-week period do not result in sensitization reactions.

5

Furthermore, hirudin is rapidly eliminated in experimental animals (half-life in the order of 1 hour) also in biologically active form via the kidneys (3).

Two other independent studies, one using dogs (5) and another (6), demonstrating the efficacy of hirudin in the prevention of DIC in rats, are in good agreement with the positive results of Markwardt et al. These researchers have recently published the first in vivo analysis of natural hirudin's effects on human hemostatic system (7).

The material shows the biological effects obtained and does not indicate toxic side effects.

15 It has also been shown that hirudin prevents DIC induced by endotoxins in pigs (8) and thus provides a possible solution to the very serious problems caused by endotoxemia, leading to a very high mortality rate in pigs.

A recent publication (9) describes intravenous and subcutaneous administration of 20 hirudin to humans. Six volunteers were used to assess the pharmacokinetics and effects on the haemostatic system of a single dose (1000 antithrombin units / kg) of hirudin. Hirudin administered intravenously has a half-life of 50 minutes and 50% of the hirudin is in active form in the urine for 24 hours after injection. An extension of the coagulation time (measured in vitro for thrombin, thromboplasticin and prothrombin) is seen as a function of the hirudin concentration in the plasma, which shows that the molecule retains its biological activity in the person's orbit. There is no change in the number of platelets, in the amount of fibrinogen or in the fibrinolytic system. Subcutaneous and intravenous injections of hirudin are well tolerated and produce no side effects. To investigate the possible occurrence of allergic reactions, 2 intracutaneous injections 30 were administered to the same individuals at a 4-week interval; no signs of sensitization were seen. In addition, no anti-hirudin antibodies are detected in the serum.

These studies suggest that hirudin may be an interesting clinical agent such as anti-coagulant. The phase of blood coagulation is not affected, taking into account the high efficacy specificity of hirudin. The anti-thrombin activity 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 DIC (3, 6), which could be expected given that DIC is accompanied by a decrease in antithrombin III (a cofactor needed for the effect of heparin) and an increase in platelet factor 4 which is a very effective antiheparin agent.

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

3 DK 174837 B1

Preparations of acellular rich extracts from leeches are commercially available as ointments (Hirucréme,

Société Nicholas, France; Exhirud-Blutgel, Plant Organ Werke, West Germany), but further tests are required with larger doses of a highly purified material to determine if this is an interesting route of administration. The preferred routes of administration are generally intravenous, intramuscular and percutaneous. Other routes of administration are described for hirudin, especially the oral route (BSM No. 3792 M).

This product can also be used in combination with other ingredients to treat psoriasis and other skin diseases of the same type, as described in DE-OS 2 101 10 393.

Hirudin can also be used as anticoagulant in clinical trials in laboratories and as a research tool. In this case, the high specificity for a unique step in blood coagulation can offer a significant advantage over the most commonly used anticoagulants, the effect of which is much less specific.

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

Hirudin's fixation activity on thrombin may also allow indirect protection of coagulation factors such as factor VIII during its purification.

Finally, the use of labeled hirudin may represent a simple and effective method for measuring the amounts of thrombin and prothrombin. In particular, labeled hirudin can be used to visualize blood clots during formation as the coagulation phenomenon results in the conversion of circulating prothrombin to thrombin at the site of formation and the labeled hirudin is fixed to the thrombin and can be visualized.

30

It is furthermore possible to envisage the direct use of transformed yeast as a drug that releases hirudin, for example by applying to the skin a cream containing this yeast which secretes hirudin.

35 In short, hirudin has a wide range of potential uses: 1) as anticoagulants for critical thrombotic disorders for prophylaxis and prevention of prevalence of preexisting thromboses; 2) as anticoagulants for reducing blood clotting and swelling after microsurgery, with 40 being widely used. live mammals, 3) as anticoagulants in extracorporeal circulatory systems, and as anticoagulants to cover synthetic biomaterials, 4) as anticoagulants in clinical tests of blood tests in laboratory tests, 5) as anticoagulants in clinical research on coagulation and as a test tool, 4 DK 6) as a possible topical agent for cutaneous application in the treatment of hemorrhoids, varicose veins and edema; 7) as a component in the treatment of psoriasis and other related disorders; and 8) finally, hirudin can be used to fix thrombin in blood storage and preparation of blood derivatives (platelets, factors VIII and IX).

As an example, hirudin can be used in therapeutic compositions at concentrations equal to 100-50,000 antithrombin units / kg / day.

Since hirudin is water-soluble, it is easy to obtain pharmaceutical compositions which are injectable or can be applied by other means using pharmaceutically acceptable bases and carriers.

Finally, it is possible to use labeled hirudin either by radioactive labeling or by a completely different type of enzymatic or fluorescent labeling performed by known techniques to do in vitro dosing or visualization in vivo, especially to visualize blood clot formation. .

A whole animal hirudin preparation was used to determine the amino acid sequence of the protein (11, 12). In the following experiments, a gene that is expressed as mes-beds RNA was cloned into the heads of fasting owls. This gene carries information for a protein (hirudin variant 2 or HV-2), whose sequence is significantly different from the sequence found throughout the animal's body (protein variant designated HV-1). There are 9 differences in amino acid residues between HV-1 and HV-2, and the differences between the two NH2 terminal 25 residues (val-val or ile-thr) may explain the obvious contradictions in the literature regarding hirudin's NH2 terminal (13 ).

FIG. Figure 1 shows the DNA sequences of the recombinant plasmid pTG717 which contain a copy of cDNA corresponding to mRNA from HV-2 as well as an amino acid sequence derived from the DNA sequence 30 at b) and the differences between that sequence and the HV-1 amino acid sequence at c).

It should be noted that the sequence of cDNA is probably incomplete and that a signal sequence may be present above the beginning of the mature protein.

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

Although the following experiments were performed with the variant HV-2, both variants are referred to hereinafter, viz. HV-i or HV-2, and optionally other variants as well as corresponding sequences 40 "hirudin" and "gene encoding hirudin", unless otherwise indicated.

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

5 DK 174837 B1 Yeast are single-celled eukaryotic organisms. The yeast genus Saccharomyces includes strains whose biochemistry and genetics have been studied extensively in laboratories; it also includes strains used in the food industry (bread, alcoholic beverages, etc.), which are thus produced in very large quantities.

5

The ease with which one can manipulate the genetics of cells from Saccharomyces ce-revisioniae, either by classical techniques or by techniques derived from the genetic methods and even better by a combination of these two types of techniques, and the long industrial history of this species makes it a preferred host in the production of foreign polypeptides.

EP-A-123 294 discloses vectors for transforming yeasts that ensure secretion of hybrid precursor peptides and allow purification of the desired polypeptide.

EP-A-116 201 describes yeast vectors containing the leader sequence from factor alpha.

EP-A-129 073 describes the expression of GRF in yeast, especially using elements of the factor alpha gene.

EP-A-123 544 discloses secretion of proteins using elements such as 20 derived from the factor alpha gene.

Therefore, the present invention relates more specifically to a yeast transformed with a functional DNA block integrated into a plasmid containing a yeast origin of replication, or integrated into a yeast chromosome containing at least 25: sequence encoding hirudin (H gene), at least one cleavage site (SC | j located immediately upstream of the H gene, a leader sequence (Lex) containing necessary elements to achieve secretion of the gene product, a DNA sequence (Str) containing signals that ensure transcription of the H gene by yeast.

35

This functional block, embedded in a plasmid or in the chromosomes of a yeast, preferably of the genus Saccharomyces, may, after transformation of the yeast, allow hirudin to be expressed, either in active form or in the form of an inactive precursor, which upon activation can regenerate hirudin.

The interesting thing about Saccharomyces cerevisiae is that this yeast is able to secrete some proteins in the culture medium; the investigation of the mechanisms responsible for this secretion is in full swing and it has been shown that, after relevant manipulations, it was possible to induce yeast to secrete human hormones that were properly formed and at all points were similar to the hormones found in human serum (14, 15).

Within the scope of the present invention, this property is utilized to achieve secretion of hirudin, providing many advantages.

First and foremost, yeast secretes few proteins, which has the advantage of succeeding in controlling the secretion of a given high-level foreign protein that a culture can be obtained in the culture medium that can constitute a large percentage of the total number of secreted 10 proteins. thus facilitating the work of purification of the desired protein.

There are several proteins or polypeptides secreted by yeast. In all the known cases, these proteins are synthesized in the form of a longer precursor, whose IMF 1 -terminal sequence is essential for its entry into the metabolic pathway leading to secretion.

15

Yeast synthesis of hybrid proteins containing the NF N terminal sequence of one of these precursors followed by the sequence of the foreign protein may in some cases lead to secretion of this foreign protein. The fact that this protein is synthesized in the form of a precursor, which is generally inactive, makes it possible to protect the cell from the possible toxic effects of the desired molecule, since the cleavage that releases the active protein occurs only in the vesicles. derived from the Golgi body and which isolates the protein from the cytoplasm.

Thus, using the metabolic pathways leading to secretion to induce the yeast to produce a foreign protein has a number of advantages: 1) it is possible to isolate a reasonably pure product in the supernatant from the culture; 2) it is possible to protecting the cell from the possible toxic effects of the mature protein; 3) on the other hand, the secreted proteins may in some cases undergo modifications 30 (glycosylation, sulfation, etc.).

Thus, the expression blocks contained in the yeast of the invention have more specifically the following structure: 35 - S (r - LgX - Sc) - H gene where LgX encodes a leader sequence necessary for the secretion of a protein corresponding to H gene; SC | is a DNA sequence encoding a cleavage site; the segment Sc | - The H gene can also be repeated several times.

As an example of the secretory system, the α-pheromone, i.e. that in the above sequence, the sequence Lex is derived from the gene for the α-sex pheromone from yeast, but other systems could be used (e.g. the system from the Killer protein) (14).

40 7 DK 174837 B1 The α-sex pheromone from yeast is a 13 amino acid peptide (enclosed in Figure 2) which is secreted into the culture medium by the yeast S. erevisiae with the mata genus type. The α-factor stops cells of the opposite sex type (Mata) in phase GI and induces biochemical and morphological changes necessary for the mating of the two types of cells. Kurjan and Herskowitz (17) have cloned the α-factor structural gene and concluded from the sequence of this gene that the α-factor of 13 amino acids was synthesized in the form of a 165 amino acid pre-precursor protein (Fig. 2). The precursor contains an amino terminal hydrophobic sequence of 22 residues (underlined with dashed line) followed by a sequence of 6110 amino acids containing 3 glycosylation sites and finally followed by 4 copies of the α-factor.

The 4 copies are separated by "spacer" sequences and the mature protein is released from the precursor by the following enzymatic activities: 1) a cathepsin B type endopeptidase, which cleaves the Lys-Arg dipeptides to COOH 15 (cleavage site shown with a bold arrow) , 2) an exopeptidase of type carboxypeptidase B which cleaves the base residues of COOH in the excised peptides; 3) a dipeptidylaminopeptidase (designated A) which removes the residues Glu-Ala and Asp-Ala.

The nucleotide sequence of this precursor further comprises 4 HindIII restriction sites shown by an arrow H.

Several fusions have been made between the gene for the α-pheromone and the mature hirudin sequence. Mata yeast cells can express these fused genes. The corresponding 25 hybrid proteins can then be processed by the signals they contain and which originate from the pre-pro sequences of the α-pheromone precursor. Therefore, one can expect that in the supernatant from the culture polypeptides with the sequence of hirudin can be isolated.

One of the constructs comprises the sequence Sc | at the 3 'end an ATG codon before the H gene; thus, the fused protein contains methionine immediately above the first amino acid of the mature hirudin sequence. After cleavage with cyanogen bromide, this polypeptide produces a hirudin molecule which can be made active after a renaturation step.

In other constructs, the cleavage signals normally used to produce the α-pheromone serve to produce in the culture supernatant polypeptides with antithrombin activity. This is the case when the sequence Sc | at its 3 'end comprises two codons which encode Lys-Arg, i. AAA or AAG with AGA or AGG; the polypeptide is cleaved by an endopeptidase which cleaves the dipeptides Lys-Arg to COOH, thus releasing hirudin.

The invention relates particularly to constructs in which the sequence before the hirudin gene encodes one of the following amino acid sequences: 1) Lys Arg Glu Ala Glu Ala Trp Leu Gin Val Asp Gly Ser Met hirudin ..., 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 5) Lys Arg hirudin ....

Of course, it is possible to use other sequences that, at the amino acid level, are selectively cleaved by an enzyme, with the proviso that this cleavage site is also not found in the hirudin itself.

10

Finally, the expression blocks after the H gene may have a yeast terminator sequence, e.g., from the PGK gene.

The expression blocks of the invention can generally be inserted into a yeast, especially Saccharomyces, either in a plasmid with autonomic replication or in the yeast chromosome.

When the plasmid is autonomous, it comprises segments that provide for its replication, viz. a replication initiation site such as the initiation site from the plasmid 2μ. In addition, the plasmid may comprise selection segments such as the gene URA3 or LEU2 which provide for complementation of the yeast strains ura3 'or Ieu2. These plasmids may additionally comprise segments which provide for their replication in bacteria when the plasmid is to be a shuttle plasmid, e.g. a replication initiation site such as the site of pBR322, a marker gene such as Ampr and / or other segments known to those skilled in the art.

The present invention also relates to yeast strains transformed with an expression block of the invention, either carried on a plasmid or integrated into its chromosomes.

Among these yeasts may be mentioned especially yeasts of the genus Saccharomyces, especially S. cere-visiae.

When the promoter is the promoter of the gene for the α-pheromone, the yeast preferably has the sex type Mata. For example, a strain of the genotype ura3 * or Ieu2 "or another, complemented by the plasmid, may be used to ensure preservation of the plasmid in the yeast at an appropriate selection pressure.

Although it is possible to prepare hirudin by fermentation of the above transformed strains in an adapted culture medium by accumulation of hirudin in the cells, it is nevertheless preferred, as can be seen from the above description, to allow hirudin to secrete in the medium, either in mature form or in the form of a precursor to be worked up in vitro.

This maturation can occur in several steps. First, it may be necessary to decouple certain segments that originate from the translation of the sequence Lex; this cleavage is performed at the level of the sequence corresponding to SC |. As stated above, before the mature 40 hirudin may be a methionine residue which is selectively cleaved with cyanogen bromide. This process can be used because the sequence encoding hirudin does not include methionine.

It is also possible to provide the N-end dipeptide Lys-Arg which cleaves to COOH 5 by a specific endopeptidase; thus, since this enzyme is active in the secretion process, the mature protein can be obtained directly in the medium. However, in some cases, it may be necessary to perform an enzymatic cleavage after secretion by adding a specific enzyme.

In some cases, especially after treatment with cyanogen bromide, it may be necessary to renaturate the protein by restoring the disulfide bridges. For this purpose, the peptide is denatured, for example, with guanidinium chlorohydrate, and then renaturated in the presence of reduced and oxidized glutathione.

Other features and advantages of the present invention will become apparent from the following examples and from the drawings, in which: Figure 1 shows the nucleotide sequence of the hirudin cDNA fragment cloned in pTG717; Figure 2 shows the nucleotide sequence of the precursor of the α-sex pheromone; 3 shows the construction of pTG834; FIG. 4 shows the construction of pTG880; FIG. 5 shows the construction of pTG882; FIG. 6 shows the construction of M13TG882; FIG. 7 shows the construction of pTG874, FIG. 8 shows the construction of pTG876; FIG. 9 shows the construction of pGT881; FIG. 10 shows the construction of pTG886; FIG. 11 shows the construction of pTG897; FIG. Figure 12 shows electrophoresis on acrylamide gel of proteins with a molecular weight of> 1000 obtained in the medium after culture of yeast transformed with pTG886 and pTG897; Figure 13 shows electrophoresis on acrylamide gel of proteins with a molecular weight of> 1000 gained in the medium after culture of yeast transformed with pTG886 and pTG897; 14 shows the construction of pTG1805; FIG. Figure 15 shows electrophoresis on acrylamide gel of proteins with a molecular weight of> 1000 obtained in the medium after culture of yeast transformed with pTG847 and pTG1805; Figure 16 shows a scheme comparing the amino acid sequences below the first cleavage site (Lys-Arg) in different constructs.

The amino acid and nucleotide sequences are not shown in the present specification so as not to make it too heavy.

EXAMPLE 1

Construction of pTG882 5

A cDNA fragment from hirudin HV-2 was extracted from a gel after cleavage of the plasmid pTG717 with PstI and then cleaved with the enzymes Hinf1 and Ahalll. The enzyme Hinfl cleaves below the first codon of the mature sequence of hirudin HV-2. The Ahalll enzyme cleaves approx. 30 base pairs after the (3 ') stop codon of the hirudin sequence. The thus-obtained HinfI * AhallI fragment was isolated on agarose gel and then eluted from this gel.

The sequence encoding the mature protein was inserted into the vector pTG880. The vector pTG880 is a derivative of the vector pTG838 (Fig. 3). Plasmid pTG838 is identical to 15 pTG833 except for the BglII site found near the transcriptional terminator of PGK.

This site was removed by filling with Klenow polymerase to give pTG838.

The plasmid pTG833 is a yeast / E. coli shuttle plasmid. This plasmid was prepared to express foreign genes in yeast. The basic segments of this vector (Fig. 3) are as follows: the gene URA3 as a selection marker in yeast, the replication initiation site from the yeast plasmid 2μ, the gene for ampicillin resistance, and the replication initiation site from the plasmid pBR322 from E. coli (these latter two segments allow for replication and selection of this plasmid in the coli bacterium), the flanking region at 5 'of the PGK yeast gene with the sequence encoding this gene, up to the SalI site, the SalI-PvuII fragment of pBR322 and the terminator of the PGK gene (20) . This plasmid is described in French patent application no.

84 07125 in the name of Transgéne S.A., filed May 9, 1984.

Plasmid pTG880 was constructed from pTG838 (Fig. 4) by inserting a short poly-linker region (originating from the bacteriophage M13) into pTG838 digested with EcoRI and Bglll, allowing a number of cloning sites to be sequenced: BglII , PstI, Hin-dlll, BamHI, SmaI and EcoRI, immediately following the 5 'region flanking the PGK yeast gene.

DNA from plasmid pTG880 was digested with BglII and SmaI, and the large fragment recovered by this cleavage was isolated and selected from a gel. The hinfl / AhaNI fragment from pTG717, which contains most of the sequence encoding hirudin HV-2, was mixed with pTG880, which was cleaved at the same time as 3 synthetic oligonucleotides (Figs.

5), which was intended to reconstitute the NH2 * terminal region from the hirudin sequence and which associates the BglII site in the vector with the Hinfl site in the fragment containing the hirudin. This mixture was subjected to ligation treatment and then served to transform E. coli cells. Plasmid pTG882 was isolated from the transformants.

40

This plasmid was used to transform yeast cells to produce hirudin under the control of the PGK promoter. However, no hirudin activity could be detected in the crude extracts of cells transformed with this vector. The reason for the absence of active hirudin production has not yet been clarified. However, the construct pTG882 served as source for a sequence encoding hirudin for the vectors for yeast secretion described below.

EXAMPLE 2

Construction of pTG886 and pTG897 First, the BglII-EcoRI fragment (230 bp) from pTG882 containing the hirudin sequence 10 was transferred to the bacteriophage M13mp8 (Fig. 6) between the BamHI and EcoRI sites. This yielded the phage M13TG882 from which an EcoRi-HindIII fragment (approximately 245 bp) could be isolated. This fragment contains the entire sequence encoding hirudin HV-2, the fusion site BamHI / Bglll, and the staple ends (HindIII-EcoRI) that allow cloning in the yeast secretion vector pTG881 (Fig. 9).

15

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

Insertion of this plasmid into E. coli makes it difficult to achieve resistance to ampicillin (and 20 other beta-lactam antibiotics). In addition, the plasmid carries the yeast genes LEU2 and URA3 expressed in strains of E. coli and of Saccharomyces. Thus, the presence of this plasmid in E. coli or in Saccharomyces allows complementation of strains lacking beta-isopropyl malate 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 with the exception of the URA3 gene, the reverse of which is constituted by the following DNA from gents (Fig. 7): 30 1) EcoRI-HindIII fragment of approx. 3.3 kb, derived from plasmid pJDB207 (18). The Hin dIII site corresponds to the coordinate 1505 of plasmid 2p, B form, and the EcoRI site to coordinate 2243. In this fragment, the LEU2 gene is inserted by polydeoxyadenilate / polydeoxy-thymidilate extension into the Pst1 site of the fragment from 2p, B-form (18).

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

3) The large EcoRI (coordinate 0) -SalI (coordinate 650) fragment from pBR322. In the PvuII site of this fragment, the 510 bp Eco RI-HindIII fragment was inserted (whose ends had already been blunted by Klenow in the presence of the 4 nucleotides) corresponding to the end of the PGK gene (20). The Eco RI end, which is cut off from the PGK gene, regenerates an Eco RI site when connected to the Pvull end of pBR322.

4) The HindIII-SalI fragment (2.15 kb) from the PGK gene (20).

12 DK 174837 B1

Plasmid pTG848 was digested with HindIII and the ends of the two thus released fragments blunted by treatment with Klenow in the presence of the 4 deoxyribonucleotides. The two fragments are ligated and, prior to transformation, this ligation mixture is subjected to treatment with HindIII, which allows the elimination of any plasmid that has retained one (or two) HindHI sites. The strain E. coli BJ5183 (pyrF) is transformed and the transformants selected for ampicillin resistance and for pyr + character. This yields plasmid pTG874 (Fig. 7), where the two HindIII sites are removed, and the orientation of the URA3 gene gives rise to transcription in the same orientation as the orientation of the phosphoglycerate kinase (PGK) gene.

The plasmid pTG874 is digested with SmaI and SalI, and a fragment of 8.6 kb is then isolated from an agarose gel. The plasmid pGT864, which comprises the EcoRI-SalI fragment (about 1.4 kb) from the gene MFal cloned in the same pBR322 sites, is digested with EcoRI. The ends of the thus-linearized plasmid are blunted by Klenow in the presence of the 4 nucleotides.

Cleavage is then performed with the enzyme SalI and the EcoRI (blunt end) SalI fragment corresponding to the gene MFal is isolated. This fragment is ligated to the Sma I-Sal I fragment (8.6 kb) of pTG874 to give the plasmid pTG876 (Fig. 8).

To remove the BglII site close to the sequence of the MFα1 promoter, a partial cleavage with BglII of the plasmid pTG876 was followed by treatment with Klenow in the presence of the 4 deoxyribonucleotides. The new plasmid obtained, pTG881 (Fig. 9), allows the 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.

25

When the fusion at the HindNI site of the foreign coding DNA allows translation in the same reading frame to be obtained, the hybrid protein obtained comprises the pre-parts of the α-pheromone.

Cloning in pTG881 of the HindIII-EcoR1 fragment carrying the hirudin gene leads to plasmid pTG886 (Fig. 10). After the cloning is performed, a BglII site is located below the fragment, which allows the fragment to be extracted in the form of Hinfl-BgIII, the Hinfl site being the same as that used above. Since BglII is unique in pTG881, it is very simple to reconstitute the 5 'end of the hirudin-coding sequence using 3 oligonucleotides that reconstitute the 5' sequence of hirudin and enable it in phase to read the pre-pro sequence of the α-pheromone followed by the mature hirudin sequence.

This new plasmid is designated pTG897 (Fig. 11).

EXAMPLE 3

Expression of hirudin in yeast 13 was used to transform a yeast TGY1sp4 (Mata, ura3 - 251 -373 - 328, his3 - 11-15) into ura + according to a technique already described (21).

A transformed colony was plated and used for inoculation of 10 mt of minimal medium plus casamino acids (0.5%). After 20 hours of culture, the cells were centrifuged and the supernatant dialyzed against distilled water and concentrated by evaporation (centrifugation in vacuo).

A culture of TGY1sp4 transformed with PTG886 and a culture of TGY1sp4 transformed with a plasmid that did not carry a hirudin sequence (TGY1sp4 pTG856) were treated in the same way in parallel. The dry precipitate was poured into 50 µl of water, and 20 µΙ boiled in the presence of 2.8% SDS and 100 mM mercaptoethanol and then applied to an acrylamide SDS gel (15% acrylamide, 0.1% SDS) ( 22). After fixation and staining with Coomassie Blue, polypeptides can be detected which accumulate in the supernatants from the cultures of TGY1sp4 / pTG886 and TGY1sp4 / pTG897 and are not present in the supernatant from the control culture (Fig. 12). In addition, these series of supplemental peptides are strongly labeled with - ^ S-cysteine, as might be expected for hirudin peptides, as this molecule is very rich in cysteine (Fig. 10).

20

The 12 and 13 electrophoresis were performed as follows:

Ten! FIG. The extracts were prepared as follows: 10 ml of minimal medium (Yeast Nitrogen Base Difco without amino acid (6.7 g / l), 10 g / l glucose enriched with 0.5% casamino acids were seeded with various strains and grown in The cells were centrifuged and the supernatant dialyzed against water (minimal retention: molecular weight 1000) and then dried by centrifugation in vacuo. The samples were then loaded with 50 μl of application buffer, of which 20 μΙ was treated as described above. , and was applied to acrylamide SDS gel (15% acrylamide, 0.1% SDS) (22).

30

The strains used are: well 2: TGY1sp4 transformed with a plasmid not containing the hirudin sequence (control), well 3: TGY1sp4 transformed with pTG886, well 4: TGY1sp4 transformed with pTG897, well 1: in well 1 markers (Pharmacia LMW kit; bottom and bottom: 94,000, 67,000, 43,000, 30,000, 20,100, 14,000).

The bands were induced by staining with Coomasie Blue R-250.

In FIG. 13, the extracts were prepared as follows: 100 ml of minimal medium + 40 µg / ml of histidine were seeded with different strains for overnight cultures. When the cell density reaches approx. 5x106 (exponential growth phase), 40 μΙ of 35 S-cysteine (9.8 mCi / ml, 1015 Ci / mmol) is added to each culture. After 10 minutes, the cells are centrifuged, placed in 10 ml complete environment (30 ° C) and incubated at 30 ° C with agitation. After 3 hours, 10 ml of the supernatant is dialyzed against water and concentrated to a volume of 0.5 ml as described for FIG.

12. Approx. 35,000 cpm (40 µl) is applied to acrylamide SDS gel (15% acrylamide, 0.1% SDS).

The protein bands are visualized after fluorography.

The strains used are: well 2: TGY1sp4 transformed with a plasmid not containing the hirudin sequence, 10 well 3: TGY1sp4 transformed with pTG886, well 4: TGY1sp4 transformed with pTG897, well 1: in well 1, markers whose molecular weight is listed (x103).

However, when the supernatants containing these polypeptides were tested for their antithrombin activity, no activity could be detected.

In the case of the polypeptide secreted by TGY1sp4 / pTG886, it is expected to undergo the normal cleavage steps of the α-pheromone precursor, yielding a hirudin molecule that is extended by 8 amino acids at its N1 - end. Therefore, it is not surprising that the polypeptide secreted by the TGY1sp4 / pTG886 cells is not active.

In contrast, in the case of the polypeptide secreted by TGY1sp4 / pTG897, the polypeptide is expected to be identical to the natural protein and thus active. Several hypotheses may explain the absence of activity: 1) Protein maturation is not complete; there may be Glu-Ala residues left at NH 2, as described for EGF (16).

2) The protein is not active, because it does not have the correct conformation, or in the culture supernatant proteins or molecules with a molecular weight of 1000 which inhibit the activity are found.

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

EXAMPLE 4 40 Activation by cleavage with cyanogen bromide

If the cause of the absence of activity is associated with the presence of additional amino acids at the NH 2 * terminal, it should be possible to restore the activity by subjecting the peptide secreted from TGYlsp4 / pTG886 to cyanogen-bromide cleavage. Namely, this reagent is specific for methionine residues, and the fused protein encoded by pTG886 contains only one methionine residue. This reaction was carried out in the following manner: yeast containing either the plasmid pTG886 or a control plasmid containing no hirudin insert is grown in 10 ml of medium for 24 hours. At this time, the culture reaches a density of 7-10x107 cells / ml. The supernatant is separated from the cells, thoroughly dialyzed against distilled water and lyophilized. The dry powder is dissolved in 1 ml of 70% formic acid, and an aliquot is used to determine the total protein content (by the Coomassie Blue staining method with the Biorad reagents); the rest of the preparation is treated with 1 ml of fresh cyanogen bromide solution (30 mg / ml) in 70% formic acid.

After eliminating oxygen with a nitrogen stream, the tubes are incubated in the dark for 4 hours at ambient temperature. All cyanogen bromide manipulations are performed with appropriate precautions and in a ventilated fume cupboard. The cleavage reaction with cyanogen-bromide is stopped by the addition of 10 volumes of distilled water and the solution is lyophilized.

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

Since the hirudin activity depends on the presence of disulfide bridges in the molecule (1), it seemed likely that the peptide digested with cyanogen bromide should be properly purified to have a biological activity. The cleaved peptides were therefore subjected to denaturation with 5M GuHCl followed by renaturation by a method known to those skilled in the art.

Briefly, the lyophilized peptides are dissolved in 400 μΙ 5M guanidinium hydrochloride (GuHCI) 30 in 250 mM Tris-HCl, pH 9.0; then the solution is made 2 mM for reduced glutathione and 0.2 mM for oxidized glutathione in a final volume of 2.0 ml (the substance concentration is 1.0 M GuHCl and 50 mM Tris).

After 16 hours of incubation at 23 ° C in the dark, the samples are dialyzed for 24 hours against 3 x 2 I 50 35 mM Tris-HCl, pH 7.5, 50 mM NaCl at 23 ° C and the final dialysate cleared by centrifugation.

Then, the antithrombin activity of the supernatants is measured. The result of this experiment (shown in Table I) clearly shows an isolation of the antithrombin activity of the supernatants from cells infected with the plasmid pTG886, while no activity is found in the control plasmids.

TABLE 1

Antithrombin activity in yeast cultures supernatants DK 174837 B1 16

Plasmid Treatment of super Activity Specific activity of the natant U / ml U / mg of initial proteins pTG886 a) after cleavage <0.3 before renaturation b) after cleavage and 2.46 102.5 renaturation control a) after cleavage <0.3 before b) after cleavage and <0.15 <5.6 renaturation

It can be concluded that yeast cells carrying a recombinant plasmid can secrete a peptide with the biological activity of hirudin after a cleavage reaction and renaturation. This suggests that the presence of additional amino acids at the NH2 terminal is sufficient to explain the absence of activity of the polypeptides present in the cultures of TGY1sp4 / pTG886, and perhaps also in the case of cultures of TGY1sp4 / pTG897 ( Ala-ha-s).

10 EXAMPLES

Insertion of a new cleavage site just before the first amino acid in the sequence encoding hirurin HV-2 - plasmid pTG1805 15 In the construct pTG897, there is a risk that the secreted peptide retains Glu-Ala tails by NH2, which would explain the absence of antithrombin activity in this material.

If this hypothesis is consistent with the reality, it should be possible to recover the activity directly in the supernatant by forming a cleavage site between the residues Glu-Ala and the first amino acid of hirudin HV-2 (isoleucine). This was done by adding a sequence encoding the duplicate LysArg, which is the recognition site for the endopeptidase implicated in the maturation of the precursor of the pheromone (Fig. 2). The construct was performed exactly as described for plasmid pTG897 except for the sequence of two synthetic oligonucleotides (Fig. 14). The resulting plasmid is designated pTG1805. The plasmid was used to transform the strain TGY1sp4 into ura +. The material secreted into the supernatant was analyzed by gel electrophoresis as described above and compared to the material recovered with TGY1sp4 / pTG897 (Fig. 15).

The strains used are: well 1: markers identical to those associated with FIG. 12 described, well 2: TGYlsp4 transformed with pTG897, well 3: TGYlsp4 transformed with pTG1805, well 17: control that does not produce hirudin.

The specific polypeptides for the strain TGY1sp4 / pTG1805 migrate more slowly than those specific for the strain TGY1sp4 / pTG897. This result suggests that the new cleavage site is not efficiently utilized by the corresponding endopeptidase. Nevertheless, the dose of hirudin in the supernatant for biological activity shows that a small proportion of this material is active in contrast to that obtained with cultures of TGY1sp4 / pTG897 which are clearly inactive (Table II).

TABLE II

Antithrombin activity in yeast cultures supernatants (10 ml)

Plasmid Specific Activity Total Activity U / mg U (10 ml) pTG856 not detectable pGT897 not detectable PTG1805 21 2 0 '15 EXAMPLE 6

New construction with a new, more efficient cleavage site allowing release of modern hirudin In the aforementioned construct (pTG897), only a little hirudin activity in the supernatant could be achieved, probably because the second cleavage site intended to release mature hirudin, only recognized with little efficiency. Therefore, a new construct was made to make this additional cleavage site more susceptible to endopeptidase by adding above a 3 amino acid sequence, Ser Leu Asp, naturally present above the first LysArg duplicate (Figs. 2 and 16).

The technique used is the same as described above except for the oligonucleotides whose sequences are shown below: 26-mer 15-mer 5 * - AGC TTCTT TGG AT AAAAG AAT T AC G τ \ ΐ AC AG AC TGC ACAG *, AGAAACCT ATTTTCTT AATGCAT AT GT CT GACGT GTCTT A, 40-Mar 18 DK 174837 B1

The amino acid sequence in the cleavage region is shown in FIG. 13. The corresponding plasmid is designated pTG1818 and is only different from pTG1805 upon insertion of the nucleotides 5'-TTG GAT AAA corresponding to the codons Ser-Leu-Asp.

The activity dosed in the supernatants of the cultures TGY1sp4 / pTG1818 according to the standard conditions already described is approx. 200 units per 10 ml of culture, ie ca. 100 times stronger than the activity found in the previous example. It should be noted that the dose can be obtained with 10 µl of non-concentrated culture medium.

EXAMPLE 7

Construction leading to synthesis of a precursor lacking the sequences Glu-Ala-Glu-Ala 15 In the two above examples, it was shown that addition of a new Lys Arg site just above the beginning of the mature hirudin sequence did it is possible to release active material into the supernatant. However, if cleavage of the precursor takes place at the first Lys Arg site (distal to the beginning of the mature sequence), then in these 20 examples in the culture medium a heavier, inactive contaminant corresponding to hirudin chains which is extended at the NH2 end. This is particularly evident in the case of TGY1sp4 / pTG1805 cultures where the inactive contaminant is in excess. This is also plausible in the case of TGY1sp4 / pTG1818 cultures, where the inactive contaminant is not in excess but could be present as described by other researchers in the case of EGF (15).

In order to avoid this loss of yield, which is the synthesis of inactive material, it was decided to make a new construction leading to the synthesis of a precursor different from the precursor synthesized by the TGY1sp4 / pTG897 cells only. , in the absence of the sequence Glu Ala Glu Ala between the cleavage site Lys Arg and the first amino acid of the mature hirudin sequence.

30 On 30 April 1985, the following strains were deposited in the Collection Nationale de Cultures de Microorganisms (CNCM) de l'Institut Pasteur, 28 rue du Docteur-Roux, Paris 15:

- TGYlsp4 pTG1818: Saccharomyces cerevisiae, strain TGYlsp4 (Mata ura3-251-373-35 328-his 3-11-15) transformed into ura + with a plasmid, pTG1818; deposit number I

441,

- TGYlsp4 pTG886: Saccharomyces cerevisiae, strain TGYlsp4 (Mata ura3-25l-373-328-his 3-11-15) transformed into ura + with a plasmid, pTG886; deposit number I

442 respectively.

40

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19 DK 174837 B1 2. Markwardt F. (1970) Methods in Enzymology, ed. Perlman G.E. and Lorand L, Academic Press, vol.19, pp. 924-932.

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Claims (16)

DK 174837 B1 A yeast transformed with a functional DNA block integrated into a piasmid containing a yeast replication initiation site, or integrated into a yeast chromosome containing at least 5: a DNA sequence encoding hirudin (H gene), at least a cleavage site (Sc | j located immediately upstream of the H gene, a leader sequence (Lex ^ containing necessary elements to achieve secretion 10 of the gene product, a DNA sequence (Sj-r) containing signals that ensure transcription of H - using yeast. Transformed yeast according to claim 1, characterized in that said functional block 15 contains at least the following sequences, counted from the 5 'end to the 3' end: a DNA sequence (S (r)) containing signals providing transcription of the Y gene in yeast is a leader sequence (Lex) containing necessary elements to achieve secretion of the gene product, a DNA sequence encoding HV, or HV2 hirudin, the sequence encoded thereof is shown in the lines ( c) respectively (b) in Fig. 1, which has the cleavage site Sc | located in front of 3. Yeast according to claim 1 or 2, characterized in that the sequence Lex is the sequence of the a-sex pheromone from yeast shown in Fig. 2. . The yeast according to claims 1-3, characterized in that the leader sequence contains prefragment of the yeast's α-sex pheromone, which is shown underlined with a dotted line in figure 2. 30 Yeast according to any one of claims 1-4, characterized in that the block contains a pre-pro sequence of α-sex pheromone at the 5 'end of the H gene, which is shown in Figure 2 and encodes amino acids 1-83. . Yeast according to any one of claims 1-5, characterized in that the H gene is followed by a yeast terminator sequence. Yeast according to claim 6, characterized in that the yeast terminator sequence is the sequence of the PGK gene. 40 Yeast according to any one of claims 1-7, characterized in that the cleavage site is the dipeptide Lys-Arg H gene, whose DNA sequence is AAA or AAG together with AGA or AGG. 21 DK 174837 B1 The yeast according to any one of claims 1-7, characterized in that the functional block is integrated into a plasmid, which contains at least one replication initiation site for yeast. 5 Yeast according to claim 9, characterized in that the site of replication is the site of replication of the plasmid 2μ. Yeast according to claim 9 or 10, characterized in that the plasmid further comprises a selection feature. A yeast according to claim 11, characterized in that the selection feature is provided by the URA3 gene. Yeast according to any one of claims 1-12, characterized in that it is a yeast of the genus Saccharomyces. Yeast according to claim 13, characterized in that it has the sex type Mata. 20 Yeast according to any one of claims 12-14, characterized in that it is a strain of S. cerevisiae. Process for producing hirudin, characterized in that a yeast according to any one of claims 1-15 is fermented in a culture medium and the hirudin produced in the culture medium is isolated. Method according to claim 16, characterized in that a) in the culture medium is fermented a yeast strain containing a functional DNA block in a plasmid or integrated into a chromosome of said yeast, which contains at least one sequence: - S {-r - Lex - SC | - H genes 35 wherein: Str is a DNA sequence comprising signals that provide for transcription of the H gene in yeast; LgX is a leader sequence that allows secretion of hirudin in mature form or secretion of hirudin in the form of a precursor for hirudin that can mature in vitro; 40 Sc | is a DNA signal encoding a cleavage site selected from ATG and sequences encoding Lys-Arg; b) the hirudin produced is isolated from the culture medium in mature form or in the form of a precursor for hirudin which can mature in vitro.