WO1991017250A1 - Procede de production recombinante d'hiridius et nouveaux hirudines - Google Patents

Procede de production recombinante d'hiridius et nouveaux hirudines Download PDF

Info

Publication number
WO1991017250A1
WO1991017250A1 PCT/EP1991/000643 EP9100643W WO9117250A1 WO 1991017250 A1 WO1991017250 A1 WO 1991017250A1 EP 9100643 W EP9100643 W EP 9100643W WO 9117250 A1 WO9117250 A1 WO 9117250A1
Authority
WO
WIPO (PCT)
Prior art keywords
hirudin
gly
glu
polypeptide
asn
Prior art date
Application number
PCT/EP1991/000643
Other languages
English (en)
Inventor
Luca Benatti
Paolo Carminati
Jacqueline Lansen
Guy Mazue
Romeo Roncucci
Paolo Sarmientos
Emanuela Scacheri
Philippe De Taxis Du Poet
Original Assignee
Farmitalia Carlo Erba S.R.L.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Farmitalia Carlo Erba S.R.L. filed Critical Farmitalia Carlo Erba S.R.L.
Priority to KR1019920700048A priority Critical patent/KR920703817A/ko
Publication of WO1991017250A1 publication Critical patent/WO1991017250A1/fr
Priority to FI920058A priority patent/FI920058A0/fi
Priority to NO92920116A priority patent/NO920116L/no

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the preparation of hirudin, which was originally isolated from the leech Hirudo 5 medicinalis. as well as to the preparation of derivatives thereof.
  • Hirudo medicinalis r is a well known and well characterize d polypeptidic inhibitor of thrombin 1 ' 2 . More particularly, it binds thrombin by ionic interactions thus preventing the cleavage of fibrinogen to fibrin and the subsequent fibrin- clot formation.
  • hirudin has demonstrated 5 efficacy in preventing venous thrombosis, vascular shunt occlusion and thrombin-induced disseminated intravascular coagulation.
  • hirudin exhibits low toxicity, little or no antigenicity and a very short clearance time from circulation 3 . 0 Three natural variants of hirudin are known.
  • HV1 The sequence of a first variant designated HV1 was determined by Dodt et al, FEBS 165 (1984) 180-184.
  • the sequence of HV1 is, according to the three letter code (Eur. J. Biochem. 13£, 9-37, 1984):
  • HV2 has been described by Dodt et al, Biol. Chem. Hoppe-Seyler 367 (1986) 803-811.
  • HV2 differs from HV1 in the following respects: l ie at position 1 instead of Val, Thr at position 2 instead of Val, Lys at position 24 instead of Gin, Asn at position 33 instead of Asp, Lys at position 35 instead of Glu, Gly at position 36 instead of Lys, Asn at position 47 instead of Lys, Glu at position 49 instead of Gin and Asn at position 53 instead of Asp.
  • a third variant designated HV3 has been described by Harvey et al, Proc. Natl. Acad. Sci. USA (1986) 1084- 1088.
  • HV3 is identical to HV2 from positions 1 to 32 and then differs from HV1 in the following respects: Gin at position 33 instead of Asp, Lys at position 35 instead of Glu, Asp at position 36 instead of Lys, Gin at position 53 instead of Asp, Pro at position 58 instead of Glu, Asp at position 62 instead of Glu, Ala at position 63 instead of Tyr (SO 3 H) , Asp at position 64 instead of Leu and Glu at position 65 instead of Gin.
  • a new approach to the preparation of hirudins and hirudin-like polypeptide has now been devised.
  • This approach is based on chemical synthesis of a nucleotide sequence encoding a hirudin or a hirudin-like polypeptide, and expression of the hirudin or hirudin-like polypeptide in recombinant organisms.
  • the cultivation of the genetically modified organisms leads to the production of the desired product displaying full biological activity.
  • the present invention provides an expression vector comprising a DNA sequence encoding a hirudin or hirudin-like polypeptide.
  • the invention further provides a host transformed with a compatible expression vector according to the invention, and also provides a synthetic DNA encoding a hirudin or hirudin-like polypeptide.
  • a DNA fragment encoding a hirudin or hirudin- like polypeptide may be single or double stranded.
  • a host in which a hirudin or hirudin-like polypeptide is able to be expressed is prepared by transforming a host with a compatible expression vector of the invention.
  • the expression vector is generally prepared by:
  • a hirudin or hirudin-like polypeptide is consequently prepared by providing a transformed host according to the invention under such conditions that a hirudin or hirudin-like polypeptide is expressed therein.
  • the hirudin or hirudin-like polypeptide can then be isolated. In this way, a hirudin or hirudin-like polypeptide may be obtained in pure form.
  • hirudin or hirudin-like polypeptide refers to hirudin in its natural HV1, HV2 and HV3 forms as well as derivatives thereof, e.g. by way of amino acid substitutions, deletions, insertions, extensions, functionalisations and chemical modifications.
  • the invention can therefore also be applied to derivatives of HV1, HV2 or HV3 having anti-thrombin activity.
  • the amino acid sequence HVl, HV2 or HV3 may be modified by one or more amino acid substitutions, insertions and/or deletions and/or by an extension at either or each end.
  • a derivative composed of such a modified sequence must of course still exhibit anti-thrombin activity.
  • the degree of homology may be 85% or more or 95% or more.
  • one or more amino acid residues of the sequence of HVl, HV2 or HV3 may be substituted or deleted or one or more additional amino acid residues may be inserted.
  • the physicochemical character of the original sequence can be preserved, i.e. in terms of charge density, hydrophobicity/hydrophilicity, size and configuration.
  • Candidate substitutions are, based on the one-letter code (Eur. J. Biochem. 138. 9-37, 1984): A for G and vice versa, V by A, L or G; K by R;
  • a short sequence of up to 50 amino acid residues may be provided at either or each terminal.
  • the sequence may have up to 30, for example up to 20 or up to 10, amino acid residues.
  • the hirudin or hirudin-like polypeptide may be subjected to one or more post-translational modification such as glycosylation, sulphation, COOH-amidation, acylation or chemical alterations of the polypeptide chain.
  • the Tyr residue at position 63 for example, may be sulphated.
  • a recombinant hirudin or hirudin-like polypeptide obtained according to the invention would not normally be sulphated at this position, unlike the natural HVl, HV2 and HV3. Further, the invention may be applied to the production of lower molecular weight derivatives which do not have the N- terminal or C-terminal portions of HVl, HV2 or HV3.
  • HVl Val-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu- Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu- Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu-Gly-Thr- Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro- Glu-Glu-Tyr-Leu-Gln
  • Hybrid HV1/HV2 (designated HV12):
  • the hirudins and hirudin-like polypeptides are prepared by recombinant DNA technology.
  • a synthetic gene encoding a hirudin or hirudin-like polypeptide is prepared.
  • the DNA coding sequence typically does not include introns.
  • the synthetic gene is inserted in an expression vector able to drive production of the recombinant product.
  • the synthetic gene is typically prepared by chemically synthesising oligonucleotides which, in total, correspond to the desired gene. The oligonucleotides are then assembled to obtain the gene.
  • a gene may therefore be constructed from four chemically synthesised oligonucleotides, each oligonucleotide representing about half of one strand of a double-stranded DNA gene.
  • the oligonucleotides are ligated and annealed to obtain the desired gene.
  • the gene sequence may be modified by site-directed mutagenesis to introduce one or more codon changes.
  • a gene is constructed with restriction sites at each end to facilitate its subsequent manipulation.
  • a preferred DNA sequence encoding HVl is shown in Figure 1 of the accompanying drawings.
  • a preferred DNA sequence encoding HV12 is shown in Figure 3. Either sequence may be modified to code for a derivative.
  • a DNA sequence may be provided which further encodes a leader peptide.
  • the leader peptide is capable of directing secretion of the hirudin or hirudin-like polypeptide from cells in which the hirudin or hirudin-like polypeptide is to be expressed.
  • the sequence encoding the leader peptide is typically fused to the 5'-end of the DNA sequence encoding the hirudin or hirudin-like polypeptide.
  • the leader peptide is preferably the OmpA leader peptide when expression in a bacterial host, such as E. coli. is required.
  • the leader peptide is preferably the leader peptide of vesicular stomatitis virus G protein (VSV G protein) where expression is to be in insect cells.
  • VSV G protein vesicular stomatitis virus G protein
  • Appropriate DNA sequences encoding the OmpA and VSV G protein leader sequences are shown in Figures 5 and 8 respectively.
  • a DNA sequence may be provided which encodes a fusion protein which is cleavable to release a hirudin or hirudin-like polypeptide.
  • a DNA sequence may be used which encodes a carrier polypeptide sequence fused via a cleavable linkage to the N-terminus of a hirudin or hirudin-like polypeptide.
  • the cleavable linkage may be one cleavable by cyanogen bromide.
  • an expression vector for expression of a hirudin or hirudin-like polypeptide, is constructed which comprises a DNA sequence encoding a hirudin or hirudin-like polypeptide and which is capable of expressing the hirudin or hirudin-like polypeptide when provided in a suitable host.
  • Appropriate transcriptional and translational control elements are provided, including a promoter for the DNA sequence, a transcriptional termination site, and translational start and stop codons.
  • the DNA sequence is provided in the correct frame such as to enable expression of the polypeptide to occur in a host compatible with the vector.
  • the expression vector typically comprises an origin of replication and, if desired, a selectable marker gene such as an antibiotic resistance gene.
  • a promoter is operably linked to the DNA sequence encoding a hirudin or hirudin-like polypeptide.
  • the expression vector may be a plasmid. In that case, preferably a promoter selected from the P-trp anc p lcc/lac promoters is operably linked to the DNA sequence.
  • the expression vector may be a virus.
  • the virus may be a recombinant baculovirus in which the polyhedrin promoter is operably linked to the DNA sequence encoding a hirudin or hirudin-like polypeptide.
  • An expression vector capable of expressing a hirudin or hirudin-like polypeptide may be prepared in any convenient fashion.
  • a DNA fragment encoding hirudin or hirudin-like polypeptide may be inserted into an appropriate restriction site of an expression vector, for example a plasmid vector.
  • a recombinant baculovirus may be prepared by:
  • step (i) cloning a gene encoding a hirudin or hirudin- like polypeptide into a baculovirus transfer vector at a restriction site downstream of the polyhedrin promoter; and (ii) co-transfecting insect cells susceptible to baculovirus infection with the recombinant transfer vector from step (i) and intact wild-type baculovirus DNA.
  • baculovirus transfer vector may be one having a unique cloning site downstream of the polyhedrin ATG start codon.
  • the product that is then expressed by the resulting recombinant baculovirus will be a fusion protein in which a N-terminal portion of the polyhedrin protein is fused to the N-terminus of a hirudin or hirudin-like polypeptide.
  • a cleavable linkage may be provided at the fusion junction.
  • the insect cells employed in step (ii) are typically Spodoptera fruqiperda cells.
  • the wild-type baculovirus is typically Auto ⁇ rapha californica nuclear polyhedrosis virus (AcNPV) .
  • An expression vector encoding a hirudin or hirudin- like polypeptide is provided in an appropriate host.
  • Cells are transformed with the gene for a hirudin or hirudin-like polypeptide.
  • a transformed host is provided under such conditions that the hirudin or hirudin-like polypeptide is expressed therein.
  • Transformed cells for example, are cultivated so as to enable expression to occur. Any compatible host-vector system may be employed.
  • the transformed host may be a prokaryotic or eukaryotic host.
  • a bacterial or yeast host may be employed, for example E. coli or S. cerevisiae. Gram positive bacteria may be employed.
  • a preferred bacterial host is a strain of E. coli type B.
  • Insect cells can alternatively be used, in which case a baculovirus expression system is appropriate.
  • the insect cells are typically Soodoptera fruqiperda cells.
  • cells of a mammalian cell line may be transformed.
  • a transgenic animal for example a non-human mammal, may be provided in which a hirudin or hirudin-like polypeptide is produced. The hirudin or hirudin-like polypeptide that is expressed may be isolated and purified.
  • the hirudin or hirudin-like polypeptide prepared according to the invention may be used in a pharmaceutical formulation, together with a pharmaceutically acceptable carrier or excipient therefor.
  • a pharmaceutical formulation is typically for intravenous administration (in which case the carrier is generally sterile saline or water of acceptable purity) .
  • the hirudin or hirudin-like polypeptide prepared according to the invention is an anti-thrombin and is suitable for treatment of thromboembolic events, such as the coagulation of blood, typically in a human patient.
  • the hirudin or hirudin-like polypeptide is coadministered with a plasminogen activator, such as tissue plasminogen activator.
  • a plasminogen activator such as tissue plasminogen activator.
  • the hirudin or hirudin-like polypeptide prepared according to the invention has been found to be compatible with the latter.
  • Figure 1 shows the nucleotide sequence of the four oligonucleotides coding for most of the hirudin HVl chain.
  • the sequence underlined indicates the Ball site which has been used for further constructions.
  • Figure shows the mode of assembling of the four oligos. Hindlll and PstI sites were included to allow subsequent manipulations.
  • Figure 2 shows the scheme of the construction of the intermediate plasmid M13-HV1, which is the source of a Ball-BamHI DNA fragment for all further bufrudin constructions.
  • Figure 3 shows the nucleotide sequence of the four oligonucleotides coding for most of the hirudin HV12 chain.
  • the sequence underlined indicates the Ball site which has been used for further constructions.
  • the lower part of the figure shows the mode of assembling of the four oligos.
  • Figure 4 shows the scheme of the construction of the intermeidate plasmid M13-HV12, which is the source of a Ball-BamHI DNA fragment for all further hirudin constructions.
  • FIG. 5 shows schematically the construction of new recombinant M13s, named OMP-HV1 and OMP-HV12, which carry respectively the complete HVl gene and the complete HV12 gene linked to the OmpA leader peptide.
  • the leader peptide sequence is underlined twice while the Ball blunt end and the Hindlll sticky end are underlined once.
  • Figure 6 shows schematically the construction of pFC-HVl and pFC-HV12 which are the plasmids used for the production of HVl and HV12 in E. coli.
  • Figure 7 shows the general structure of the plasmid pOMP-HVl used for the production of hirudin in E. coli.
  • Figure 8 shows the nucleotide sequence and assembling of the synthetic oligos used for the secretion of hirudin from insect cells.
  • the sequence underlined indicates the VSV G protein leader peptide.
  • FIG 9 is a schematic representation of the construction of a new recombinant M13, named VSV-HV12, where the complete bufrudin gene is linked to the VSV G protein leader peptide.
  • Figure 10 shows schematically the construction of pAc-HV12 which has been used as a transfer vector to the baculovirus genome.
  • pAcYMl is the starting plasmid widely used as an acceptor of heterologous sequences to be transferred to the virus.
  • Figure 11 shows the nucleotide sequence and assembling of the synthetic oligos coding for the beginning of the hirudin chain. The ATG codon coding for the additional methionine residue is underlined.
  • Figure 12 shows schematically the construction of pAcFTl which has been used for intracellular hirudin expression.
  • Figure 13 is a schematic representation of the new transfer plasmids, named pAcFTl-HVl and pAcFTl-HV12, which carry respectively the complete HVl and HV12 sequences linked to the first 18 amino acids of polyhedrin. These plasmids have been used to transfer the heterologous sequence to the baculovirus genome.
  • the nucleotide coding sequences were designed on the basis of the Escherichia coli preferred codons 4 .
  • a Ball restriction site was engineered very close to the 5' end of the synthetic genes to allow insertion of the coding sequences in different expression vectors. Indeed, the same synthetic genes were used for expression in bacterial and insect cells. In the case of insect cells methods were developed which yielded secreted or cytoplasmic products.
  • the recombinant plasmid M13-HV1 was used as the source of the HVl gene for all the expression vectors used in the Examples.
  • a HVl2 gene was synthesised and assembled in the same way.
  • the oligos used to assemble the gene are shown in Figure 3.
  • Oligos 3 and 4 code for different amino acids than oligos 3 and 4 of Figure 1.
  • Example 2 Expression and secretion of hirudin from E. coli cells
  • leader peptide responsible for an efficient secretion must be present at the NH 2 end of HVl or HV12 7 ' 8 . This extra sequence is then cleaved off. In vivo, during secretion, by a specific E. coli leader peptidase, yielding the correct mature sequence 9 .
  • FIG. 5 Their sequence, shown in Fig. 5, includes also the beginning of the HVl gene coding for the first 10 amino acids.
  • the presence of the Ball site allowed the joining of this synthetic piece to the rest of the HVl coding sequence while the presence of the upstream Hindlll site allowed the joining to the M13 vector.
  • the synthetic Hindlll-Ball fragment was ligated to a Ball-BamHI piece from M13-HV1 and inserted in M13mpl8, obtaining a new plasmid named OMP-HV1.
  • the schematic representation of this new plasmid construction is also shown in Figure 5. Equivalent manipulations starting from M13-HV12 gave OMP-HV12 ( Figure 5).
  • the hirudin gene can be excised as a Hindlll-BamHI fragment which codes for the OmpA Shine- Dalgarno and leader peptide followed by the hirudin coding sequence.
  • This restriction fragment is now ready to be inserted in an appropriate expression vector.
  • Several expression systems could, theoretically, be employed to obtain high level production of heterologous proteins in bacteria. The system based on the promoter Ptrp ⁇ as bee used with success in our laboratory in the past 13 . Again, even in the case of the selected promoter, the levels of expression of a given polypeptide cannot be predicted. The use of the promoter P rp ° r t * ⁇ e expression of hirudin has never been reported to date.
  • Plasmid pFC33 shown in Figure 6, has already been described in the literature 13 . It carries the resistance to the antibiotic ampicillin and the bacterial promoter Ptrp which drives expression of proapolipoprotein Al. Following digestion of pFC33 with Hindlll and BamHI, the large Hindlll-BamHI fragment, carrying the antibiotic resistance gene and the promoter, was isolated and joined to the Hindlll-BamHI fragment from OMP-HV1 or from OMP-HV12 coding for the hirudin HVl or the hirudin derivative HV12. The details of this construction are shown in Figure 6. We isolated new plasmids, named pFC-HVl and pFC-HV12, which are the final plasmids for the production of HVl and HV12 in E. coli.
  • a main object of the present invention is the use of E. coli strains of the type B for the expression and secretion to the periplasm of hirudin and its derivatives. Indeed, we have found that insertion of plasmid pFC-HVl in type B strains of the bacterium E. coli brings high level production of hirudin. Interestingly, different strain types of E. coli do not work as efficiently and it seems, therefore, that the host strain type is crucial for the successful production of hirudins.
  • telomere encoding a hirudin or hirudin-like polypeptide are available and can be used for the production of a hirudin or hirudin-like polypeptide.
  • Preferred strains are ATCC 12407, ATCC 11303, NCTC 10537.
  • Competent cells of strain NCTC 10537 were prepared using the calcium chloride procedure of Mandel and Higa 14 . Approximately 200 ⁇ l of a preparation of these cells at 1 x 10 9 cells per milliliter were transformed with 2 ⁇ l of plasmid DNA (approximate concentration 5 ⁇ g/ml) . Transformants were selected on plates of L-agar containing lOO ⁇ g/ml ampicillin. Two small colonies were streaked with wooden tooth picks (each as three streaks about 1 cm long) onto L-agar containing the same antibiotic.
  • hirudin activity was present at the level of about 50 ⁇ g/ml. This activity was absent in control periplasmic fractions.
  • productivity of the hirudin variant HV12 was equivalent to 80 ⁇ g/ml.
  • Example 3 Anticoagulant activity of recombinant HVl obtained from E. coli
  • the anticoagulant activity of the hirudin variant HVl was also tested in an activated partial thromboplastin time (aPTT) test and in a thrombin time (T.T.) test. Both tests were performed using an automatic coagulometer (ACL300 Research, Instrumentation Laboratory, Milan, Italy) . To normal citrated human plasma, were added increasing concentrations of the recombinantly produced hirudin HVl. The samples were then assayed with the automatic coagulometer which permits determination of aPTT and T.T. by adding automatically to the plasma the appropriate reagents and recording the rate of formation of the clot.
  • aPTT activated partial thromboplastin time
  • T.T. thrombin time
  • aPTT was determined using cephaline and calcium chloride (automated APTT reagent, General Diagnostic, USA) and T.T. was determined using human thrombin (Fibrindex, Ortho Diagnostic, Milan, Italy) at a concentration of 5IU/ml.
  • the reagents were prepared, stored and used according to the manufacturer's instructions.
  • the clotting times obtained in the aPTT and T.T. tests were plotted against the concentrations of the recombinant protein. In each test, the concentration which doubled clotting times relative to a normal plasma was calculated. The value obtained for the aPTT test was 210 ng/ml, whereas for the T.T. test the value was 90 ng/ml.
  • VSV-HV12 The synthetic Hindlll-Ball fragment was joined to a purified Ball-BamHI fragment from M13-HV12 carrying the HV12 gene and inserted in M13mpl8 previously cut with Hindlll and BamHI.
  • This construction which yielded a new plasmid named VSV-HV12 is schematically shown in Figure 9. From VSV-HV12 we have excised a BamHI-BamHI DNA fragment carrying the HV12 gene fused to the VSV leader peptide which was then inserted into the vector pAcYMl 18 , as shown in Figure 10. The resulting plasmid was named pAc-HV12.
  • the VSV-HV12 coding sequence must be transferred to the baculovirus genome under the transcriptional control of the polyhedrin promoter.
  • insect cells For this purpose, we co-transfected insect cells with a wild-type baculovirus DNA and with the transfer vector pAc-HV12. As insect cells, Spodoptera frugiperda cells were chosen as host cells. Experimental details are as follows: S. frugiperda cells were transfected with a mixture of infectious AcNPV DNA and plasmid DNA representing the individual recombinant transfer vectors by a modification of the procedure described by Summers et al 19 .
  • One microgram of viral DNA was mixed with 25-100 ⁇ g of plasmid DNA and precipitated with (final concentrations) 0.125 M calcium chloride in the presence of 20 mM HEPES buffer, pH 7.5, 1 mM disodium hydrogen orthophosphate, 5mM potassium chloride, 140 mM sodium chloride and 10 mM glucose (total volume 1ml) .
  • the DNA suspension was inoculated onto a monolayer of 10 6 S. fruqiperda cells in a 35-mm tissue culture dish, allowed to adsorb to the cells for 1 h at room temperature, then replaced with 1 ml of medium. After incubation at 28°c for 3 days the supernatant fluids were harvested and used to produce plaques in S.
  • Plaques containing recombinant virus were identified by their lack of polyhedra when examined by light microscopy. Virus from such plaques was recovered and after further plaque purification was used to produce polyhedrin-negative virus stocks.
  • Example 5 Expression of HVl and HV12 in the cytoplasm of insect cells
  • Hirudin and its derivatives could also be produced and accumulated in the cytoplasm of S. fruqiperda cells.
  • HVl and HV12 are based on the expression of a fusion polypeptide where the first 18 amino acids of polyhedrin are joined in frame to the 65 amino acids of HVl or HV12.
  • the presence of the NH 2 end sequence of polyhedrin allows high level expression 20 .
  • polyhedrin portion and the HVl or HV12 sequence we put a methionine residue which allows the release of the HVl or HV12 moiety by treatment of the hybrid protein with CNBr.
  • the recombinant baculoviruses were obtained as described in Example 4. Infection of S. fruqiperda cells was carried out according to standard procedures 19 . Cultivation of infected insect cells lead to the cytoplasmic accumulation of the fusion protein. This hybrid protein was the source of recombinant HVl or HV12.
  • Several methods are available from the literature which can be used to cleave the hybrid with CNBr 21 ' 22 .
  • the application of the method of Olson et al 22 has allowed us to obtain the HVl and HV12 of the correct polypeptide sequences. These two molecules displayed anti-thrombin activity.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Public Health (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hematology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Diabetes (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

On prépare un polypeptide d'hirudine ou de la famille des hirudines possédant une activité anti-thrombine, au moyen de méthodologies d'ADN recombiné. Le polypeptide d'hirudine ou de la famille des hirudines est exprimé, de préférence, dans E. coli ou des cellules d'insectes. Le polypeptide de la famille des hirudines est, de préférence, une protéine hybride provenant de HV1 et HV2, qui possède la séquence d'aminés suivante: HV12: Val-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu-Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu-Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu-Gly-Thr-Pro-Asn-Pro-Glu-Ser-His-Asn-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln, la séquence soulignée représentant la partie HV2.
PCT/EP1991/000643 1990-05-10 1991-04-05 Procede de production recombinante d'hiridius et nouveaux hirudines WO1991017250A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
KR1019920700048A KR920703817A (ko) 1990-05-10 1991-04-05 히루딘 및 히루딘-형 폴리펩타이드의 재조합적 제조방법
FI920058A FI920058A0 (fi) 1990-05-10 1992-01-07 Foerfarande foer produktion av hirudiner och hirudin-liknande polypeptider med rekombinantteknologi.
NO92920116A NO920116L (no) 1990-05-10 1992-01-09 Fremgangsmaate ved rekombinant fremstilling av hirudiner og hirudinlignende polypeptider

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9010552.9 1990-05-10
GB909010552A GB9010552D0 (en) 1990-05-10 1990-05-10 Method for the recombinant production of hirudins and novel hirudins

Publications (1)

Publication Number Publication Date
WO1991017250A1 true WO1991017250A1 (fr) 1991-11-14

Family

ID=10675801

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1991/000643 WO1991017250A1 (fr) 1990-05-10 1991-04-05 Procede de production recombinante d'hiridius et nouveaux hirudines

Country Status (16)

Country Link
EP (1) EP0482144A1 (fr)
JP (1) JPH05500615A (fr)
KR (1) KR920703817A (fr)
CN (1) CN1057294A (fr)
AU (1) AU7652591A (fr)
CA (1) CA2063575A1 (fr)
CS (1) CS133191A3 (fr)
FI (1) FI920058A0 (fr)
GB (1) GB9010552D0 (fr)
HU (1) HUT59961A (fr)
IE (1) IE911578A1 (fr)
IL (1) IL98074A0 (fr)
NO (1) NO920116L (fr)
PT (1) PT97604A (fr)
WO (1) WO1991017250A1 (fr)
ZA (1) ZA913503B (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0576792A3 (fr) * 1992-04-09 1994-08-24 Biogal Gyogyszergyar
US5516656A (en) * 1990-11-08 1996-05-14 Nippon Mining Company, Limited Production of a new hirudin analog and anticoagulant pharmaceutical composition containing the same
US5972648A (en) * 1993-09-28 1999-10-26 Japan Energy Corporation Hirudin analogs, methods of manufacture thereof and anticoagulant compositions having these as active ingredients
WO2012101181A1 (fr) 2011-01-25 2012-08-02 Université Catholique de Louvain Compositions et procédés de greffe de cellules
WO2013110354A1 (fr) 2012-01-25 2013-08-01 Université Catholique de Louvain Compositions et procédés de transplantation de cellules
CN115572329A (zh) * 2021-06-21 2023-01-06 王大勇 一组活性增强代谢较慢的菲牛蛭基因重组水蛭素及其制备方法

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102005002978B4 (de) * 2005-01-21 2013-04-25 Merz Pharma Gmbh & Co. Kgaa Rekombinante Expression von Proteinen in einer disulfidverbrückten, zweikettigen Form
CN108220369B (zh) * 2017-12-04 2021-03-12 广东双骏生物科技有限公司 一种生产重组水蛭素的方法
CN115677850B (zh) * 2021-07-24 2024-03-08 王大勇 具备较强抗凝血活性的医蛭基因突变水蛭素及其制备方法
CN114736289B (zh) * 2022-03-17 2023-07-18 华南理工大学 一种带有酪氨酸硫酸化修饰的水蛭素的化学合成方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986003517A1 (fr) * 1984-12-13 1986-06-19 Gen-Bio-Tec Gesellschaft Für Gentechnologische Und Sequence d'adn codant une proteine semblable a l'hirudin et procede de preparation de cette proteine
EP0352228A2 (fr) * 1988-07-19 1990-01-24 Ciba-Geigy Ag Protéines
EP0171024B1 (fr) * 1984-08-10 1991-09-11 Hoechst Aktiengesellschaft Procédé à génie génétique pour la production de hirudines et moyens pour la mise en oeuvre de ce procédé

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0171024B1 (fr) * 1984-08-10 1991-09-11 Hoechst Aktiengesellschaft Procédé à génie génétique pour la production de hirudines et moyens pour la mise en oeuvre de ce procédé
WO1986003517A1 (fr) * 1984-12-13 1986-06-19 Gen-Bio-Tec Gesellschaft Für Gentechnologische Und Sequence d'adn codant une proteine semblable a l'hirudin et procede de preparation de cette proteine
EP0352228A2 (fr) * 1988-07-19 1990-01-24 Ciba-Geigy Ag Protéines

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5516656A (en) * 1990-11-08 1996-05-14 Nippon Mining Company, Limited Production of a new hirudin analog and anticoagulant pharmaceutical composition containing the same
US5573929A (en) * 1990-11-08 1996-11-12 Japan Energy Corporation Secretion vector for hirudin or hirudin analog production
EP0576792A3 (fr) * 1992-04-09 1994-08-24 Biogal Gyogyszergyar
US5972648A (en) * 1993-09-28 1999-10-26 Japan Energy Corporation Hirudin analogs, methods of manufacture thereof and anticoagulant compositions having these as active ingredients
WO2012101181A1 (fr) 2011-01-25 2012-08-02 Université Catholique de Louvain Compositions et procédés de greffe de cellules
WO2013110354A1 (fr) 2012-01-25 2013-08-01 Université Catholique de Louvain Compositions et procédés de transplantation de cellules
CN115572329A (zh) * 2021-06-21 2023-01-06 王大勇 一组活性增强代谢较慢的菲牛蛭基因重组水蛭素及其制备方法
CN115572329B (zh) * 2021-06-21 2024-02-06 王大勇 一组活性增强代谢较慢的菲牛蛭基因重组水蛭素及其制备方法

Also Published As

Publication number Publication date
NO920116D0 (no) 1992-01-09
JPH05500615A (ja) 1993-02-12
HUT59961A (en) 1992-07-28
KR920703817A (ko) 1992-12-18
IL98074A0 (en) 1992-06-21
CS133191A3 (en) 1992-01-15
IE911578A1 (en) 1991-11-20
ZA913503B (en) 1992-02-26
PT97604A (pt) 1992-02-28
CA2063575A1 (fr) 1991-11-11
EP0482144A1 (fr) 1992-04-29
CN1057294A (zh) 1991-12-25
FI920058A0 (fi) 1992-01-07
AU7652591A (en) 1991-11-27
HU9200088D0 (en) 1992-06-29
NO920116L (no) 1992-03-10
GB9010552D0 (en) 1990-07-04

Similar Documents

Publication Publication Date Title
AU710535B2 (en) Chimeric proteins and muteins of tissue factor pathway inhibitors TFPI and TFPI-2
AU650880B2 (en) Oxidation resistant thrombomodulin analogs
EP0501821B1 (fr) Polypeptides antithrombiniques
US5227469A (en) Platelet aggregation inhibitors from the leech
WO1991017250A1 (fr) Procede de production recombinante d'hiridius et nouveaux hirudines
AU646633B2 (en) Soluble analogs of thrombomodulin
Benatti et al. Secretion of biologically active leech hirudin from baculovirus-infected insect cells
DE69329083T2 (de) Halbleiteranordnung
EP0710285B1 (fr) Analogues d'un polypeptide antithrombine et leur procede de preparation
CA2150909A1 (fr) Anticoagulant extrait de la salive de protostomia
RU2131462C1 (ru) Фрагмент днк, рекомбинантный полипептид с антитромбиновой активностью, способ его получения (варианты), фармацевтическая композиция, рекомбинантный вектор (варианты)
du Poet et al. Production of the HV1 variant of hirudin by recombinant DNA methodology
US20020069421A1 (en) Large scale expression and purification of recombinant proteins

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA FI HU JP KR NO SU US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE

WWE Wipo information: entry into national phase

Ref document number: 920058

Country of ref document: FI

WWE Wipo information: entry into national phase

Ref document number: 2063575

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 1991907785

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1991907785

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1991907785

Country of ref document: EP