NO302375B1 - Yeast for the preparation of hirudin and a process for the preparation of hirudin - 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

The present invention relates to vectors for the expression

of the DNA sequence which codes for a hirudin or hirudin analogues, the secretion of the hirudin into the culture medium of yeast strains transformed by these vectors and methods by which an active hirudin can be obtained by fermentation.

The anticoagulant activity present in the salivary glands

in medicinal leeches, Hirudo medicinalis, originates

in a small polypeptide known as hirudin (1). This very special and very effective inhibitor of thrombin has been the subject of extensive research in recent times, as it is potentially a very useful therapeutic agent. Nevertheless, the great difficulties and costs associated with isolation and purification have prevented it from achieving wider application and has even ruled out the possibility that it could be examined from a clinical standpoint.

The possibility of producing hirudin by cloning genes and expressing them using recombinant DNA technology has already been demonstrated by the cloning of a natural leech gene that codes for hirudin and expression in the microorganism E. coli (applicants' French patent application no. 84/04,755 filed March 27, 1984). Although it has been possible to produce in E. coli a peptide that has biological activity, it is very important to produce hirudin in other types of microorganisms. The clinical application of hirudin actually requires the product to be of very high purity, and the removal of pyrogenic impurities can give rise to problems with regard to the purification of hirudin from extracts of E. coli bacteria.

Furthermore, the hirudin synthesized by E. coli remains intracellular and must therefore be purified from a very large number of E. coli peptides. For these reasons it was useful to effect

that the hirudin gene was expressed in yeast, which does not produce substances that are pyrogenic or toxic to humans, and which can secrete proteins into the culture medium.

It is only now that one begins to understand the mechanism of action

of hirudin as a. anticoagulant. The substrate for the binding of hirudin is thrombin, which is a proteolytic enzyme which, upon activation (activated by factor X) from its zymogen form, prothrombin, cleaves fibrinogen in the circulation and converts it to fibrin, which is necessary for the formation of the blood clot. The dissociation constant for the 1:1 thrombin/hirudin complex (0.8 x 10~<1>(^) suggests an extremely strong association between these molecules (2). In practice, the non-covalent complex between these two molecules can be considered to be indissociable in vivo.

Hirudin is a highly specific inhibitor of thrombin with a

much higher affinity than the natural substrate fibrinogen. Furthermore, it is not necessary for other coagulation factors or other plasma constituents to be present. The specific and very significant antithrombin activity of hirudin makes it obvious that it can be used clinically as an anticoagulant.

Hirudin has been studied extensively in animals

due to its anticoagulant properties. The most. detailed study (3) describes the activity of hirudin in the prevention of venous thrombosis, clogging of blood vessels and disseminated intra-vascular coagulation (DIC) in rats. Hirudin is well tolerated by rats, dogs, rabbits and mice when it is present in a very pure form and injected intravenously. LD^q in mice is greater than 500,000 U/kg body weight (ie 60 mg/kg). Another survey (4) shows that. mice tolerate doses of up to 1 g/kg, and that rabbits tolerate up to 10 mg/kg both intravenously and subcutaneously. In mice, repeated injections over a period of two weeks do not lead to sensitization reactions.

Moreover, hirudin in experimental animals is quickly eliminated (half-life of the order of 1 hour) while it is still in a biologically active form, via the kidneys (3).

Two other independent investigations, one using dogs (5) and the other (6) showing the activity of hirudin in the prevention of DIC in rats, are in agreement with the positive results obtained by Markwardt and his co-workers. These researchers have recently published the first in vivo analysis of the effects of native hirudin on the human hemostatic system (7). The subject exhibited the expected biological effects without any sign of toxic side effects.

It was also possible to show that hirudin prevents an endotoxin-induced DIC in pigs (8) and thus constitutes a potential solution to the very serious problem caused by endotoxemia which leads to high mortality in pigs.

A single very recent publication (7) describes the intravenous and subcutaneous administration of hirudin to humans. Six volunteers were used to evaluate the pharmacokinetics and effects on the hemostatic system of a single dose (1000 AT-U/kg) of hirudin. When given intravenously, hirudin has a half-life of 50 min, and 50% of hirudin appears in an active form in the urine within 24 hours after injection. One

prolongation of the coagulation time can be observed (measured in vitro for thrombin, thromboplastin and prothrombin) according to the concentration of hirudin in the plasma, which shows that the molecule retains its biological activity in the test subject's circulation. No change was observed in the number of platelets in the fibrinogen level or in the fibrinolytic system. As in intravenous injections, subcutaneous injections of hirudin are well tolerated and cause no side effects.

To test for the possible appearance of allergic reactions, two intracutaneous injections were given to the same test subjects at an interval of four weeks. No signs of sensitization were observed. Moreover, no anti-hirudin antibodies were detected in the serum.

These investigations suggest that hirudin may constitute a clinical agent useful as an anticoagulant. As a result of the high specificity of the action of hirudin, the pre-phase of blood coagulation is not affected. The antithrombin activity is dose-dependent, and the action of hirudin is rapidly reversible as a result of its rapid renal elimination. It has been possible to show that hirudin is far superior to heparin for the treatment of DIC (3, 6), as might be expected in light of the fact that DIC is accompanied by a decrease in antithrombin III (a cofactor necessary for the action of heparin )

and a release of blood factor 4 which is a very effective antiheparin agent.

An investigation has shown the possibility that hirudin can be absorbed by human skin (9), although the results obtained are somewhat difficult to interpret.

Commercially available preparations of acellular crude extracts from leeches are available as an ointment (Hirucréme, Société Nicholas, France, Exhirud-Blutgel, Plantorgan Werke, West Germany), but further trials with larger doses of highly pure material are necessary to bring about clear whether this is a favorable supply route. In general, the preferred routes of administration are the intravenous and intramuscular routes, and the percutaneous route. Other routes of administration have been reported for hirudin, particularly the oral route (BSM no. 3,792 M).

In combination with other components, this product can also be used in the treatment of psoriasis and other skin conditions of the same type, as described in DOS 2 101 393.

Hirudin can also be used as an anticoagulant

in clinical laboratory trials and as a research tool. In this case, the high specificity for a single step can

in the coagulation of blood have a significant advantage compared to the most frequently used anticoagulants, whose effect is much less specific.

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

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

Finally, the use of labeled hirudin can constitute a simple and effective method for measuring thrombin and prothrombin levels. More specifically, labeled hirudin can be used to visualize blood clots (clots) in the formation process, as the phenomenon of coagulation includes the conversion of circulating prothrombin to thrombin at the site of formation, as the labeled hirudin is bound to thrombin and has the ability to be made visible.

It is also possible to imagine the direct use of transformed yeasts as a drug that releases hirudin, e.g. by spreading a cream containing the aforementioned yeasts that excrete hirudin on the skin.

It is therefore the purpose of the present invention to produce hirudin using yeast.

As a summary, hirudin produced according to the invention has a number of possible applications: 1) as an anticoagulant in critical thrombosis situations, for prophylaxis and to prevent the expansion of existing thrombi; 2) as an anticoagulant for the reduction of tumor-like blood accumulations (haematomas) and swelling after microsurgery, in which situations significant use is made of live leeches; 3) as an anticoagulant in extracorporeal circulation systems and as an anticoagulant for coating synthetic biomaterials; 4) as an anticoagulant in clinical tests on blood samples in laboratory experiments; 5) as an anticoagulant in clinical research on coagulation, and as an experimental tool; 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 diseases; 8) finally, hirudin can be used for the binding of thrombin during the storage of blood and the production of blood derivatives (platelets, factors VIII and IX).

As a guide, hirudin can be used in therapeutic mixtures at concentrations corresponding to 100-50,000 antithrombin U/kg per day.

As hirudin is soluble in water, it is easy to obtain injectable pharmaceutical mixtures or pharmaceutical mixtures which can be used via other routes using pharmaceutically acceptable carriers and auxiliaries.

Finally, it is possible to use hirudin labeled either with

a radioactive marker or with any type of enzymatic or fluorescent marker, the labeling being carried out by known techniques, to carry out in vitro investigations or in vivo imaging, in particular for the visualization of blood clot formation.

A preparation of hirudin from the whole animal has been used to determine the amino acid sequence of the protein (10, 11). In the experiments that follow, a gene has been cloned that is expressed as a messenger RNA in the heads of leeches that have been subjected to fasting. This gene carries information for a protein (hirudin variant 2 or HV-2), whose sequence is significantly different from that found throughout the animal's body (protein variant known as HV-1). There are 9 differences in amino acid residues between HV-1 and HV-2, and the differences between the two t^-terminal residues (Val-val or Ile-Thr) may explain the apparent contradictions in the literature regarding the 1S^-terminal end of hirudin ( 12).

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

It should be noted that the cDNA sequence is likely to be incomplete and that there may be a signal sequence upstream of the beginning of the mature protein.

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

Although the experiments set forth below were performed with the HV-2 variant, in the following, unless otherwise indicated, "hirudin" and "gene encoding hirudin" shall be used to refer to both variants, i.e., HV-1 or HV-2, and similarly for other possible variants and the corresponding sequences.

One of the purposes of the present invention is to produce hirudin by yeast.

Yeasts are unicellular eukaryotic organisms: the Saccharomyces genus of yeasts includes strains whose biochemistry and genetics are the subject of extensive research' in the laboratory.

It also includes strains used in the food industry

(bread, alcoholic beverages and the like) which are consequently produced in very large quantities.

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

For this reason, the present invention specifically relates to a yeast that allows the production of hirudin, characterized in that it contains at least: - a DNA sequence that codes for hirudin, one of its variants or a precursor for hirudin (gene H); - a leader sequence (Lex) containing the necessary elements to achieve secretion of the product from gene H; - a DNA sequence (Str) which contains the signals that induce transk3{Q2i3i7 5 of gene H in the yeast; - a DNA sequence (Scl) which codes for the amino acids ser-leu-asp in connection with the endopeptidase recognition sequence lys-arg immediately upstream of the gene H, and

- optionally a DNA sequence that codes for a selection property.

When it is integrated in a plasmid or in the chromosomes of a yeast, preferably of the genus Saccharomyces, this functional block after transformation of said yeast can make it possible to express hirudin either in an active form or in the form of an inactive precursor which is capable of regenerating hirudin when activated.

The benefit of Saccharomyces cerevisiae lies in the fact that this yeast

can secrete certain proteins into the culture medium, great progress is being made in the investigation of the mechanisms responsible for this secretion, and it has been shown that it was possible, after the right manipulations, to get the yeast to secrete properly treated human hormones that were in all respects similar to those found in human serum (13, 14).

In connection with the present invention, this property is used to achieve excretion of hirudin, as this offers many advantages.

Firstly, yeast secretes few proteins, which has the advantage, if it is possible to direct the secretion of a given foreign protein at a high level, that it allows the achievement of a product in the culture medium that can represent a high percentage of the total secreted proteins , and consequently facilitates the work of producing the desired protein in a pure state.

Different proteins or polypeptides are secreted by yeast.

In all known cases, these proteins are synthesized in the form of a longer precursor, the N1^-terminal sequence which is decisive for entering the metabolic pathway leading to excretion.

The synthesis in yeast of hybrid proteins containing the NE^-terminal sequence of one of these precursors followed by the sequence of the foreign protein can in certain cases lead to the secretion of this foreign protein. The fact that this foreign protein is synthesized in the form of a precursor which is consequently generally inactive enables the cell to be protected against the possible toxic effects of the molecules sought, as the cleavage that releases the active protein only takes place in small vesicles (vesicles) obtained from the Golgi apparatus, which isolates the protein from the cytoplasm.

The use of the metabolic pathways that lead to the excretion of

getting the yeast to produce a foreign protein therefore has a number of advantages:

1) it allows a relatively pure product to be recovered from the culture supernatant, 2) it allows the cells to be protected against the possible toxic effects of the mature protein, 3) moreover, the secreted proteins can in certain cases undergo modifications (glycosylation, sulphation and the like) ).

For this reason, the expression blocks according to the invention will more specifically have the following structure:

S,. — L — S , — H gene.

tr ex cl ^

Lex codes for a leader sequence that is necessary for secretion of the protein

corresponding to the H gene; Scler a DNA sequence that codes for a cleavage

seat, and further the element Scl-H gene can be repeated several times. S^. contains the signals that carry out the transcription of gene H in yeast.

As an example of a secretion system, that of alpha-pheromone was chosen, i.e. in the sequence indicated above, L originates from the gene for the alpha-sex pheromone of yeast, but other systems can be used (e.g. the Killer protein system) (13 ).

The alpha sex pheromone of yeast is a peptide consisting of 13 amino acids (shown boxed in Fig. 2) which is secreted into the culture medium of S. cerevisiae yeast with MATa sex. The alpha factor stops the cells of the opposite sex type (MATa)

in phase G1 and induces biochemical and morphological changes that are necessary for pairing of the two types of cells. Kurjan and Herskowitz (16) have cloned the structural gene for the α-factor and have drawn the conclusion from the sequence of this gene that this 13-amino acid α-factor was synthesized in the form of a 165-amino acid precursor preproprotein (Fig. 2). The precursor contains an amino-terminal hydrophobic sequence of 22 residues (dotted underline) followed by a sequence of 61 amino acids containing 3 glycosylation sites, and finally followed by 4 copies of the α-factor. The 4 copies are separated by "spacers"

("spacer") sequences and the mature protein is released from the precursor as a result of the following enzyme activities: 1) a cathepsin-B-type endopeptidase that cuts at the COOH side of Lys-Arg dipeptides (cleavage site indicated by

a broad arrow),

2) an exopeptidase of the carboxypeptidase B type that cuts off the basic residues present at the COOH end of the excised peptides, 3) a dipeptidyl aminopeptidase (known as A) that removes Glu-Ala- and Asp-Ala - the leftovers.

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

Several fusions were performed between the α-pheromone gene and

the mature hirudin sequence. Yeast cells of the MATa type can express these fused genes. The corresponding hybrid proteins can then be processed as a result of the signals they contain, which originate in the prepro sequences of the α-pheromone precursor. It is therefore expected that polypeptides having the hirudin sequence will be recovered in the culture supernatant.

In one of these constructs, the Scl, sequence contains an ATG codon at the 3' end preceding the H gene, and the fused protein consequently contains a methionine immediately upstream of the first amino acid of the mature hirudin sequence. After cleavage with cyanogen bromide, this polypeptide gives a hirudin molecule that can be made active after a renaturation step.

In other constructions, the cleavage signals which are normally used for the production of the α--f eromone serve to produce in the culture growth medium polypeptides which possess antithrombin activity. This is the case when the sequence contains two codons that code for Lys-Arg, i.e. AAA or AAG with AGA or AGG, at its 3' end, and the polypeptide is cut with an endopeptidase that cuts off Lys-Arg dipeptides on the COOH side and thereby releasing hirudin.

More specifically, the invention relates to the constructions where the sequence that precedes the hirudin gene codes for one of the following amino acid sequences:

1) Lys Arg Glu Ala Glu Ala Trp Leu Gin Val Asp Gly Ser

With 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 Ala Ser Leu Asp Lys Arg hirudin or

5) Light Arg hirudin...

It is of course possible to imagine other sequences such as

at the amino acid level is selectively cut with an enzyme, with the proviso that this cleavage site is not also present in hirudin itself.

Finally, the expression blocks may have, after the H gene, a yeast termination sequence, e.g. that of the PGK gene.

In general, the expression blocks according to the invention can be integrated into a yeast, especially Saccharomyces, either in an autonomous

replicating plasmid or in the goat chromosome.

When the plasmid is autonomous, it will contain elements that ensure its replication, i.e. a replication initiation point such as that of the 2 u plasmid. In addition, the plasmid may contain selection elements such as the URA3 or LEU2 gene, which ensure complementation of ura3~ or leu2~ yeast strains. These plasmids can also contain elements that ensure their replication in bacteria when the plasmid must be a "shuttle" plasmid, e.g. an origin of replication such as that of pBR322, a marker gene such as Amp<r>and/or other elements known to those skilled in the art.

The present invention relates to yeast strains transformed by an expression block, either carried by a plasmid or integrated into its chromosomes. Among these yeasts are yeasts of the genus Saccharomyces, and in particular

S. cerevisiae must be specially mentioned.

When the promoter is that of the α-pheromone gene, the yeast will preferably be of sex type MATa. For example, a strain of genotype ura3 or leu2~ or the like, complemented by the plasmid to ensure maintenance of the plasmid in the yeast by applying a correct selection pressure, will be used.

Although it is possible to produce hirudin by fermentation

of the above transformed strains in a suitable culture medium by accumulation of hirudin in the cells, it is nevertheless preferred, as appears from the above description, to cause the hirudin to be secreted into the medium, either in a mature form or in the form of a precursor that must be processed in vitro.

This maturation can be carried out in several stages. First, it may be necessary to cleave certain elements originating from the translation of the sequence L , and this cleavage will be performed on the sequence corresponding to Sc^. As stated above,

can there be a methionine that precedes the mature hirudin

selectively cleaved by cyanogen bromide. This method can be used because the sequence that codes for hirudin does not include methionine.

It is also possible to obtain at the N-terminal end, the Lys-Arg dipeptide which is cut on the COOH side by a specific endopeptidase, as this enzyme is active in the secretion process. It is therefore possible in this way to obtain the mature protein directly in the medium. In some cases, however, it may be necessary to provide enzymatic cleavage after secretion, by adding a special enzyme.

In some cases, especially after treatment with cyanogen bromide, it may be necessary to renature the protein by re-forming the disulphide bridges. For this purpose, the peptide is denatured, e.g. with guanidine hydrochloride and then renatured in the presence of reduced and oxidized glutathione.

Other special features and advantages of the present invention will become apparent by reviewing the examples below.

The accompanying figures show the following:

Fig. 1 shows the nucleotide sequence of the cDNA fragment of hirudin

cloned in pTG 717,

fig. 2 shows the nucleotide sequence of the alpha-sex-pheromone-for-runner,

fig. 3 schematically shows the construction of pTG834,

fig. 4 schematically shows the construction of pTG880,

fig. 5 schematically shows the construction of pTG882,

fig. 6 schematically shows the construction of the M13TG882,

fig. 7 schematically shows the construction of pTG874,

fig. 8 schematically shows the construction of pTG876,

fig. 9 schematically shows the construction of pTG881,

fig. 10 schematically shows the construction of pTG886,

fig. 11 schematically shows the construction of pTG897,

fig. 12 shows an acrylamide gel electrophoresis of the proteins

with MV (molecular weight) > 1,000 which is obtained in the medium after cultivation of yeast strains transformed by pTG886 and pTG897, fig. 13 shows an acrylamide gel electrophoresis of the proteins

with MV > 1,000, which is obtained in the medium after cultivation of

yeast strains transformed with pTG886 and pTG897,

fig. 14 schematically shows the construction of pTG1805,

fig. 15 shows an acrylamide gel electrophoresis of proteins with MV > 1,000, which are obtained in the medium after cultivation of yeast strains

transformed with pTG847 and pTG1805,

fig. 16 is a diagram comparing the amino acid sequences

on the underside of the first split-wing seat (Light-Arg)

in different constructions.

The amino acid and nucleotide sequences are not repeated in the present description so as not to burden the latter unnecessarily, but they form an explicit part thereof.

Example 1 - Construction of pTG822

A cDNA fragment of hirudin HV-2 was extracted from a gel after digestion of plasmid pTG717 with Pstl, and then digested. at the same time with the enzymes Hinfl and Ahalll. The enzyme Hinfl cuts downstream of the first codon of the mature hirudin HV-2 sequence. The enzyme Ahalll cuts approx. 30 base pairs behind (3') the stop codon of the hirudin sequence. The HinfI-AhaIII fragment thus obtained was isolated on agarose gel and then eluted from this gel.

The sequence coding for the mature protein was introduced into vector pTG880. Vector pTG880 is a derivative of vector pTG838 (Fig. 3). Plasmid pTG838 is identical to pTG833 except for the BglII site located near the PGK transcription terminator. This site has been eliminated by filling in using Klenow polymerase to give pTG838.

Plasmid pTG833 is a yeast/E. coli shuttle plasmid. This plasmid was constructed for the expression of foreign genes in yeast. The basic elements of this vector (Fig. 3) are as follows: the URA3 gene as a selection marker in yeast, the origin of replication of the gjasr-2u plasmid, the gene for resistance to ampicillin and the origin of replication of the E. coli plasmid pBR322 (these latter two elements enabling the plasmid to be propagated and selected in colibacill), the 5'-flanking region of the yeast PGK gene with the sequence coding for this gene up to the SalI site, the SalI-PvuII fragment of pBR322 and the PGK gene terminator ( 19). This plasmid has already been described in applicants' French Patent Application No. 84/07,125, filed on May 9, 1984.

Plasmid pTG880 was constructed from pTG838 (Fig. 4) by inserting a short polylinker region (derived from a bacteriophage M13) into pTG838 cut with EcoRI and BglII, which allows

a series of cloning sites to be arranged in the order Bglll, Pstl, Hindlll, BamHI, SmaI and EcoRI just after the 5<1> region flanking the yeast PGK gene. DNA of plasmid pTG880 was digested with BglII and SmaI, and the large fragment obtained as a result of this digestion was isolated and eluted from a gel. The HinfI-AhaIII fragment of pTG717 containing most of the sequence encoding hirudin HV-2 was mixed with digested pTG880 along with 3 synthetic oligonucleotides (Fig. 5) intended to reconstitute the NH2-terminal region of the hirudin sequence , the nucleotides reconnecting the Bglll site of the vector to the HinfI site of the fragment containing hirudin. This mixture was subjected to a ligase-. treatment and then it served to transform

E. coli cells. Plasmid pTG882 was recovered in transformants. -

This plasmid served to transform yeast cells in order to obtain hirudin under the control of the PGK promoters. However, no hirudin activity was observed in the crude extracts of cells transformed with this vector. The reason for the lack of active hirudin production is still not clear. However, construct pTG882 served as a source of the hirudin coding sequence for the yeast secretion vectors described below.

Example 2 - Construction of pTG886 and pTG897

First, the BglII-EcoRI fragment (230 bp) of pTG882 containing the hirudin sequence was transferred into bacteriophage M13mp8

(Fig. 6) between the BamHI and EcoRI sites. This resulted in phage M13TG882, from which it was possible to isolate an EcoRI-HindIII fragment (about 245 bp). This fragment contains the entire coding sequence for hirudin HV-2, the BamHI/BglII fusion site and cohesive ends (HindIII-EcoRI) allowing it to be cloned into the yeast secretion vector pTG881 (Fig. 9).

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

The introduction of this plasmid into E. coli allows resistance to ampicillin (and other B-lactam antibiotics) to be achieved. In addition, this plasmid carries the yeast genes LEU2 and URA3, which are expressed in strains of E. coli and Saccharomyces. The presence of this plasmid in E. coli or in Saccharomyces consequently allows the achievement of complementation of strains deficient in 8-isopropylmalate dehydrogenase or OMP decarboxylase.

Plasmid pTG881 is constructed as follows:

The starting plasmid is pTG848 (identical to pTG849 described in

French Patent Application No. 83/15 716, with the exception of the ura3 gene, whose direction is reversed), and it consists of the following DNA fragments (Fig. 7):

1) The approx. 3.3 kb EcoRI-HindIII fragment derived from plasmid pJDB207 (17). The HindIII site corresponds to coordinate 105 of the 2u plasmid, B-form, the EcoRI site to coordinate 2243. In this fragment, the LEU2 gene has been inserted by polydeoxy-adenylate/polydeoxythymidylate extension into the Pstl site of B-form 2u- the fragment (17); 2) the HindIII fragment of the -URA3 gene (18); 3) The large fragment of EcoRI (coordinate 0)-SalI (coordinate 650) of pBR322. In the PvuII site of this fragment, the fragment EcoRI-Hindlll of 510 base pairs has been inserted (whose ends have previously been blunted by the action of the Klenow enzyme in the presence of the 4 nucleotides) corresponding to the end of the PGK gene (19). When added to the PvuII end of pBR322, the corrected EcoRI end of the PGK gene regenerates an EcoRI site; 4) The HindIII-SalI fragment (2.15 kb) of the PGK gene (19).

Plasmid pTG848 cut with HindIII and the ends of the two fragments which

thereby released is blunted by treatment with Klenow enzyme in the presence of the 4 deoxyribonucleotides. Ligation of the two fragments is carried out and, prior to transformation, this ligation mixture is subjected to the action of HindIII, which allows the removal of any plasmid form which has retained a HindIII site (or two such sites). E. coli strain BJ5183 (pyrF) is transformed and the transformants are selected for resistance to ampicillin and for the pyr<+> property. Plasmid pTG874 is thereby obtained (Fig. 7), where the two HindIII sites have been eliminated, and the orientation of the URA3 gene gives rise to transcription in the same direction as that of the phosphoglycerate kinase (PGK) gene.

Plasmid pTG874 is cut with Smal and SalI, and the 8.2 kb fragment is then isolated from an agarose gel. The plasmid pTG864, which comprises the EcoRI-Sall fragment (about 1.4 kb) of the MFa1 gene cloned in the same sites of pBR322, is cut with EcoRI. The ends of the plasmid which have been linearized in this way are blunted by the action of Klenow enzyme in the presence of the 4 nucleotides. The digestion is then carried out with the enzyme SalI, and the EcoRI (butt end) SalI fragment corresponding to the MFa1 gene is isolated. This latter fragment is ligated with the Smal-Sall fragment (8.2 kb) of pTG874, yielding plasmid pTG876 (Fig. 8).

To eliminate the proximal BglII site of the MFa1 promoter sequence, partial digestion was made of plasmid pTG876 with BglII followed by the action of Klenow enzyme 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 MFal gene and the EglII site of the end

of the PGK gene.

When fusion of the HindIII site of the foreign coding DNA allows translation to be achieved in the same reading phase, the resulting hybrid protein comprises the preproportions of the α-pheromone.

Cloning into pTG881 of the HindIII-EcoRI fragment carrying the hirudin gene leads to plasmid pTG886 (Fig. 10). Once the cloning has been performed, there is a Bglll site on the underside of the fragment, and this allows the fragment to be extracted again in a Hinfl-Bglll form, the Hinfl site being the same as that which was

used above. As there is only a single BglII site in pTG881, it is very easy to reconstitute the 5' end of the sequence coding for hirudin using 3 oligonucleotides that reconstitute the 5' sequence of hirudin and allow the prepro sequence of the apheromone followed by the mature hirudin sequence to be read in phase.

The new plasmid is known as pTG897 (Fig. 11).

Example 3 - Expression of hirudin .- i . yeast

DNA of pTG897 was used to transform a TGY1sp4 yeast (MATa, ura3 - 251 - 373 - 328, his3 - 11-15) into ura + according to a technique already described (20).

A transformed colony was further cultivated (subcultured) and used for inoculation of

10 ml minimum medium plus casamino acids (0.5%). After 20 hours of cultivation, the cells were centrifuged and the growth medium was dialyzed against distilled water and concentrated by evaporation (centrifugation under vacuum).

In parallel, a TGY1sp4 culture transformed with pTG886 and a TGY1sp4 culture transformed with a plasmid that did not carry the sequence for hirudin (TGY1sp4/pTG856) were treated in the same way. The dry pellets were taken up in 50 pl of water,

and 20 µl was boiled in the presence of 2.8% SDS and 100 mM mercapto-

ethanol and then deposited on an acrylamide-SDS gel (15% acrylamide, 0.1% SDS) (21). After fixation and staining with Coomassie blue, polypeptides can be detected, which are collected in the supernatants of the culture TGY1sp4/pTG886 and TGY1sp4/pTG897, and which are absent in the growth medium of co-

the equation culture (fig. 12). Moreover, these series of additional peptides are strongly labeled with [ "^S]cysteine, as can be expected for hirudin peptides, as this molecule is very rich in cysteine (Fig. 10).

The electrophoresis patterns shown in fig. 12 and 13 were produced

as follows:

For fig. 12, the extracts were prepared as follows: 10 ml minimum medium (yeast-nitrogen base Difco without amino acid,

(6.7 g/l) and glucose (10 g/l), supplemented with 0.5% casamino acids were inoculated with different strains and grown for 20 h (stationary phase). The cells were centrifuged and the supernatant dialyzed against water (retention minimum: MV 1000) and then dried by centrifugation under vacuum. The samples were then taken up with 50 µl of loading buffer, of which 20

were processed as described above, and deposited on acrylamide-SDS gel (15% acrylamide, 0.1% SDS) (21).

Strains used are:

- well 2: TGY1sp4 transformed with a plasmid not containing the hirudin sequence (comparison); - well 3: TGY1sp4 transformed with pTG886; - well 4: TGY1sp4 transformed with pTG897; - well 1: the reference markers were deposited in well 1 (Pharmacia LMW kit from top to bottom: 94,000, 67,000, 43,000, 30,000, 20,100, 14,000).

The bands are made visible by staining with Coomassie blue R-250.

For fig. 13, the extracts were prepared as follows: 100 ml minimum medium + 40 µg/ml histidine were inoculated with different strains for overnight cultivation. When the cell density reaches approx. 6 35

5 x 10 (exponential growth phase), becomes 40 yl S cysteine

(9.8 mCi/ml, 1.015 Ci/mmol) added to each culture. After 10 min, the cells are centrifuged, taken up in 10 ml of complete medium (30°C) and incubated at 30°C with agitation. After 3 hours, 10 ml of the supernatant liquid is dialysed against water and concentrated to a volume of 0.5 ml, as described for fig. 12. Approx. 35,000 cpm (40 µl) are deposited on acrylamide-SDS gel (15% acrylamide, 0.1% SDS). The protein bands are made visible after fluorography.

The strains used are:

- well 2: TGY1sp4 transformed with a plasmid that does not • contain the hirudin sequence;

- well 3: TGY1sp4 transformed with pTG886,

- well 4: TGY1sp4 transformed with pTG897; - well 1: in well 1 markers were deposited which had the specified molecular weight (x10<3>).

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

As for the polypeptide secreted by TGY1sp4/pTG886,

this is expected to undergo the normal steps of cleavage of the α-pheromone precursor, which would yield a hirudin molecule extended by 8 amino acids at its 1S 2 -terminal end. It is therefore not surprising that the polypeptides secreted by the TGY1sp4/pTG886 cells are not active.

In contrast, it is expected for a polypeptide secreted

of TGY1sp4/pTG897 that this is identical to the natural protein and therefore active. Several hypotheses can explain the lack of activity:

1) Maturation of the protein is not complete, Glu-Ala residues are possibly back at the NH2 end, as has been described for EGF (14). 2) The protein is inactive because it does not have the correct conformation or alternatively because there are floating proteins or molecules with a molecular weight of 1,000 in the culture which prevent the activity. 3) The protein is incomplete because it has undergone intracellular and/or extracellular proteolysis.

Example 4 - Activation by cleavage with cyanogen bromide

If the reason for the lack of activity is connected with the presence of additional amino acids at the Nt^-terminal end, it should be possible to recover the activity by subjecting the peptide secreted by TGY1sp4/pTG886 to cleavage with cyanogen bromide. This reagent is in fact specific for

methionine residues and the fusion protein encoded by pTG886 contains only a single methionine. This reaction was carried out as follows: yeast containing either plasmid pTG886 or a comparison plasmid which does not contain a hirudin insert is grown in 10 ml of medium for 24 hours. On this

time the culture reaches a density of 7-10 x 10 7 cells.ml<-1>The growth medium is separated from the cells, dialyzed

intensively against distilled water and then lyophilized. The dry powder is dissolved in 1 ml of formic acid with a strength of

70% and one portion is used for determining the total protein content (according to the methods for staining with Coomassie blue with the reagents sold by Biorad); the rest of the preparation being treated with 1 ml of new cyanogen bromide solution (30 mg/ml) in formic acid with a strength of 70%.

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

The cleaved peptides are redissolved in 10 ml of distilled water and relyophilized twice. Finally, the peptides are dissolved in a small volume of distilled water, and a portion is used to measure the antithrombin activity. The rest of the sample is lyophilized and subjected to the renaturation steps described below.

As hirudin activity depends on the presence of disulfide bridges

in molecule (1), it seems likely that the peptide cleaved with cyanogen bromide must be properly renatured to show biological activity. The cleaved peptides were therefore subjected to denaturation in 5 M GuHCl, followed by renaturation

according to a method known to those skilled in the art.

In summary, the lyophilized peptides are dissolved

in 400 µl 5 M guanidinium chloride (GuHCl) in 250 mM Tris.HCl,

pH 9.0; the solution is then made 2 mM in reduced glutathione, and 0.2 mM in oxidized glutathione in a final volume of 2.0 ml (the final concentration is 1.0 M GuHCl and 50 mM Tris).

After 16 hours of incubation at 23°C in the dark, the samples are dialyzed for 24 hours against 3 times 2 1 50 mM Tris.HCl, pH 7.5, 50 mM NaCl at 23°C, and the final dialysate is clarified by centrifugation.

The antithrombin activity of the supernatants is then measured. The result of this experiment, shown in Table I, clearly shows that there is a recovery of the antithrombin activity in the supernatants of the cells infected with plasmid pTG886, while there is no activity with the comparison plasmid.

In conclusion, yeast cells carrying a recombinant plasmid can secrete a peptide having the biological activity of hirudin, after a cleavage and renaturation reaction. This shows that the presence of additional amino acids at the NH2-terminal end will be sufficient to explain the lack of activity of the polypeptides present in the TGY1sp4/pTG886 cultures, and possibly also in the case of the TGY1sp4/pTG897 cultures (Glu- Ala tails).

Example 5 - Introduction of a new split wing seat immediately

before the first - amino acid in the sequence coding for hirudin HV- 2 - plasmid pTG1 805

In the construction of pTG897, there is a risk that the secreted peptide will retain its Glu-Ala tails at the NH2 end, which would explain the lack of antithrombin activity in this material. If this hypothesis corresponds to the actual situation, it should be possible to recover the activity directly in the supernatant liquid by forming a cleavage site between the Glu-Ala residues and the first amino acid of hirudin-HV-2 (iso-leucine). This was done by adding a sequence that codes for the Lys-Arg doublet, which is the recognition site for the endopeptidase involved in the maturation of the pheromone precursor (Fig. 2). The construction was obtained in exactly the same way as described for plasmid pTG897, with the exception of the sequence of two synthetic oligonucleotides (Fig.

14). The resulting plasmid is designated pTG1805. The plasmid was used to transform strain TGY1sp4 into ura. The material separated in the supernatant was analyzed by gel electrophoresis as described above and compared to the material obtained with TGY1sp4/pTG897 (Fig. 15).

The strains used are:

- well 1: markers identical to those in fig. 12,

- well 2: TGYlsp4 transformed with pTG897,

- well 3: TGY1sp4 transformed with pTG1805,

- well 4: comparison sample which does not produce hirudin.

The polypeptides specific for strain TGY1sp4/pTG1805 migrate more slowly than those specific for strain TGY1sp4/pTG897. This result suggests that the new cleavage site is not efficiently used by the corresponding endopeptidase. Nevertheless, the analysis of hirudin shows the growth medium

on the basis of its biological activity that a small part

of this material is active, in contrast to that obtained with cultures of TGY1sp4/pTG897, where the material is clearly inactive (Table II).

Example 6 - Novel design comprising a new, more efficient cleavage site, allowing release of mature hirudin

In the above construct (pTG897), only a small amount of hirudin activity was obtained in the supernatant, probably because the second cleavage site engineered to release the mature hirudin is recognized inefficiently. A new construction was therefore carried out, calculated to make this additional cleavage site more accessible to the endopeptidase, by adding to it on the upstream side a sequence of the 3 amino acids Ser Leu Asp which naturally occur on the upstream side of the first Lys-Arg- doublet (Figs. 2 and 16).

The technique used is the same as that described above, except for the oligonucleotides, the sequences of which are as follows:

The amino acid sequence in the cleavage region is shown in fig. 16.

The corresponding plasmid is designated pTG1818, and differs

from pTG1 805 only by the insertion of the nucleotides 5 '-TCTTTG GAT, which correspond to the codons Ser-Leu-Asp.

The activity measured in the supernatants of the TGY1sp4/pTG1818 culture, following the standard conditions already described, is approx. 200 units per 10 ml culture, equivalent to approx. 100 times greater than that found in the previous example. It should be noted that the measurement can be performed with 10 ml of unconcentrated culture medium.

Example 7 - Construction leading to the synthesis of a precursor

which is free of Glu-Ala-Glu-Ala sequences

In the previous two examples it was shown that the addition of

a new Lys-Arg site immediately upstream of the beginning of the mature hirudin sequence allowed active material to be released into the supernatant. In these examples, although cleavage of the precursor takes place at the first Lys-Arg site (distant with respect to the beginning of the mature sequence), however, a heavier, inactive contaminant corresponding to hirudin chains extended at the NE^ terminus can be obtained in the culture medium. This is particularly obvious in the case of TGY1sp4/

pTG1805 cultures, where the inactive contaminant predominates. This also remains likely in the case of TGY1sp4/pTG1818 cultures, where the inactive contamination does not predominate but may be present, as described by others in the case of EGF (14). To avoid this loss in yield represented by the synthesis of inactive material, it was decided to undertake a new construction leading to the synthesis of a precursor that differs only from that synthesized by TGY1sp4/pTG897 cells in the absence of Glu Ala Glu Ala- the sequence between the Lys-Arg cleavage site and the first amino acid of the mature hirudin sequence.

The following strains were deposited at the Collection Nationale

de Cultures de Microorganismes (CNCM) (National Collection of Microorganism Cultures) at the INSTITUT PASTEUR, 28 rue du Docteur-Roux, PARIS 15éme, 30 April 1985:

TGY1sp4/pTG1818: Saccharomyces cerevisiae strain

TGY1sp4 (MATa ura3-251-373-328-his 3-11-15) transformed into ura by a plasmid pTG1818; deposit no. In 441.

TGY1sp4/pTG886: Saccharomyces cerevisiae strain

TGY1sp4 (MATaura3-251-373-328-his 3-11-15) transformed into ura+ by a plasmid pTG886; deposit no. In 442.

Claims (12)

1. Yeast for the production of hirudin, transformed with a functional DNA block, integrated into a plasmid containing an origin of replication from yeast, characterized in that said block contains at least; - a DNA sequence encoding hirudin, one of its variants or a precursor of hirudin (gene H); - a leader sequence (Lex) containing the necessary elements to achieve secretion of the product from gene H; - a DNA sequence (S^) containing the signals that carry out the transcription of gene H in the yeast; - a DNA sequence (Scl) which codes for the amino acids ser-leu-asp in connection with the endopeptidase recognition sequence lys-arg immediately upstream of the gene H, and -, optionally a DNA sequence which codes for a selection property. 2. Yeast as stated in claim 1, characterized in that said DNA sequence (Scl) codes for the amino acid sequence lys-arg-glu-ala-glu-ala-ser-leu-asp in connection with the endopeptidase recognition sequence lys-arg immediately upstream of gene H. 3. Yeast as specified in one of claims 1 or 2, characterized in that the sequence Lex is the leader sequence for the yeast ct sex pheromone. 4. Yeast as specified in one of claims 1-3, characterized in that the gene H is followed by a termination sequence from yeast. 5. Yeast as specified in claim 4, characterized in that the termination sequence from yeast is that of the gene PGK. 6. Yeast as stated in claims 1-5, characterized in that the origin of replication is that of the plasmid 2\ x. 7. Yeast as specified in one of claims 1-6, characterized in that the plasmid also contains a selection's own chap. 8. Yeast as stated in claim 7, characterized in that the selection property is provided by the gene URA3. 9. Yeast as specified in one of claims 1-8, characterized in that it is a yeast of the genus Saccharomyces. 10. Yeast as specified in claim 9, characterized in that it has the gender type Matalpha. 11. Yeast as specified in claims 9-10, characterized in that it belongs to a strain of S . cerevisiae. 12. Method for producing hirudin by fermentation of a yeast as stated in one of claims 1-11 in a culture medium and recovery of the hirudin product in the culture medium in the form of mature hirudin, characterized by; a) a strain of yeast containing a functional DNA block, carried by a plasmid containing at least; - a DNA sequence that codes for hirudin, one of its variants or a precursor of hirudin (gene H), - a leader sequence (Lex) that contains the necessary elements to achieve secretion of the product from gene H; - a DNA sequence (Stf) containing the signals that carry out the transcription from gene H in the yeast; - a DNA sequence (Scl) that codes for the amino acids Ser Leu Asp in conjunction with the endopeptidase recognition sequence Lys Arg immediately upstream of the gene H is grown in a growth medium; and - optionally a DNA sequence that codes for a selection property. b) the hirudin product is recovered in the culture medium in the form of mature hirudin.. V