WO1999029838A1 - Fibrinogen-converting enzyme hybrids - Google Patents
Fibrinogen-converting enzyme hybrids Download PDFInfo
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- WO1999029838A1 WO1999029838A1 PCT/US1998/026086 US9826086W WO9929838A1 WO 1999029838 A1 WO1999029838 A1 WO 1999029838A1 US 9826086 W US9826086 W US 9826086W WO 9929838 A1 WO9929838 A1 WO 9929838A1
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
- C12N9/6418—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals from snakes
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- 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
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention is directed to multidomain proteins made up of a first polypeptide that is a fibrinogen-converting enzyme or a snake venom-derived proteinase and a second polypeptide that is a member of a binding pair, where the other member of the binding pair can be used to remove the fusion protein from a fibrin preparation that was formed through the action of the converting enzyme.
- the invention is further directed to recombinant methods of forming the multidomain protein, to nucleic acids and vectors used in such methods, and to methods of forming fibrin using the fusion protein.
- the invention further provides a fusion protein between a fibrinogen-converting enzyme and a polypeptide designed to facilitate covalent attachment of one member of a binding pair.
- the invention provides for an aggregate of (a) the first polypeptide which is covalently linked to a member of a binding pair and (b) the second polypeptide which binds to the first by way of its binding the member of a binding pair.
- Fibrin sealants are widely used to reduce bleeding in surgery and to seal blood vessels and tissues that have been dissected either in surgery or through wounding.
- the term “fibrin” can be viewed as a misnomer in this context since historically "fibrin” sealants have been delivered as a material containing the precursor of fibrin, namely fibrinogen.
- fibrinogen material has been co-delivered at the site to be sealed with a proteinase enzyme that converts the fibrinogen to fibrin. Once a sufficient amount of fibrin is formed from the fibrinogen, the fibrin spontaneously polymerizes into a fibrin polymer which — when sufficient polymer is assembled — forms a fibrin clot.
- the conversion enzyme has been bovine-derived thrombin. Recently, however, an effective sealant has been described that delivers fibrin, in a form that is prevented from polymerizating, to the site that is to be sealed. At the site, the polymerization prevention conditions are reversed, and an effective clot forms. See, Edwardson et al., European Patent Application No. EP 592,242. As described in EP 592,242, the fibrin in the sealant can be formed by contacting fibrinogen with a fibrinogen converting enzyme that is bound to a solid support. The solid support allows for the removal of the converting enzyme from the sealant.
- this fibrin sealant of EP 592,242 can be an autologous sealant that is rapidly prepared from a small amount of a patient's blood only minutes before surgery, and this preparation can be done using standard laboratory equipment. Processes for deriving the fibrinogen material of prior art sealants are much more demanding and more difficult to automate. Specialized tools for preparing fibrin have also recently been described, and these tools allow an autologous sealant to be prepared from a patient in a rapid, highly reproducible, highly reliable, and highly safe manner.
- the invention provides a multidomain protein comprising: a first polypeptide chain comprising a fibrinogen-converting enzyme; and a second polypeptide chain comprising a first member of a binding pair, wherein the second polypeptide chain is linked to the first polypeptide chain (1) directly by bonds utilizing the N-terminal amino groups, the C-terminal carboxy groups, or side-chain functionalities, (2) via a bifunctional linkage moiety linking said groups or functionalities, or (3) by the first member binding to the second member of the binding pair, wherein the second member of the binding pair is covalently attached to the first polypeptide chain.
- the multidomain protein is a recombinant protein comprising a continuous amino acid sequence that includes the second polypeptide chain and the first polypeptide chain, and the second polypeptide chain comprises a polypeptide with biotin-binding activity.
- the second polypeptide chain can comprise a multivalent binding entity such as an antibody, Streptavidin or avidin with two or more binding sites.
- a multivalent binding entity can bind to a first polypeptide via a ligand for the binding sites which is covalently linked to the first polypeptide, leaving at least one other binding site available to bind another molecule of ligand.
- the invention provides a nucleic acid encoding a recombinant fusion protein comprising a continuous amino acid sequence which comprises: a first polypeptide chain comprising a fibrinogen-converting enzyme; and a second polypeptide chain comprising a member of a binding pair, wherein the first and second polypeptides are either fused directly via a peptide bond or fused via a linker polypeptide chain.
- the invention provides a recombinant fusion protein comprising a contiguous polypeptide chain comprising: a first polypeptide comprising a fibrinogen-converting enzyme; and a second polypeptide comprising a two or more of (a) amino acid side chains that can be used to attach a binding partner or (b) O-linked or N- linked polysaccharrides that can be used to attach a binding partner.
- the amino acid residues of the second polypeptide are selected to minimize the amount of secondary structure forming adjacent to said amino acids with attachable side chains
- the invention provides a method of preparing a fibrin composition, the method comprising: (1) contacting a composition comprising fibrinogen with an enzyme effective to convert fibrinogen to fibrin, forming a fibrin composition, wherein said enzyme comprises a multidomain protein comprising a first polypeptide chain comprising a fibrinogen-converting enzyme, and a second polypeptide chain comprising a first member of a binding pair, wherein the second polypeptide chain is linked to the first polypeptide chain (a) directly by bonds utilizing the N-terminal amino groups, the C-terminal carboxy groups, or side-chain functionalities, (b) via a bifiinctional linkage moiety linking said groups or functionalities, or (c) by the first member binding to the second member of the binding pair, wherein the second member of the binding pair is covalently attached to the first polypeptide
- the method further comprises: (2) forming a monomeric fibrin composition from the fibrin composition.
- the method further comprises: (3) contacting the monomeric fibrin composition with a solid support having bound thereto a second member of the binding pair effective to bind the first member.
- the method further comprises after the contacting of step (3): (4) removing the solid support and recovering a resulting monomeric fibrin composition.
- the invention provides a conjugate protein comprising an alpha polypeptide chain comprising a snake venom-derived proteinase effective to convert prothrombin to thrombin, and a second molecule that is a member of a binding pair covalently attached to the alpha polypeptide chain.
- the invention provides a nucleic acid encoding a recombinant fusion protein comprising a continuous amino acid sequence comprising: an alpha polypeptide chain comprising a snake venom-derived proteinase effective to convert prothrombin to thrombin; and a beta polypeptide chain comprising a member of a binding pair, wherein the alpha and beta polypeptides are fused directly via a peptide bond or fused via a linker polypeptide chain.
- the invention provides a method of preparing a thrombin composition, the method comprising: (1) contacting a composition comprising prothrombin with a snake-derived enzyme effective to convert to prothrombin to thrombin, thereby forming a thrombin composition, wherein said enzyme comprises a multidomain protein comprising an alpha polypeptide chain comprising the enzyme, and a beta polypeptide chain comprising a first member of a binding pair, wherein the beta polypeptide chain is linked to the alpha polypeptide chain (a) directly by bonds utilizing the N-terminal amino groups, the C-terminal carboxy groups, or side-chain functionalities, (b) via a bifunctional linkage moiety linking said groups or functionalities, or (c) by the first member binding to the second member of the binding pair, wherein the second member of the binding pair is covalently attached to the first polypeptide chain.
- the method further comprises: (2) contacting the thrombin composition with a solid support having bound thereto a second member of the binding pair effective to bind the first member.
- the method further comprises: (3) removing the solid support and thereby recovering a resulting thrombin composition.
- a bifunctional linkage group is a molecule having two sites for attaching to a protein or polypeptide.
- Preferred bifunctional linkage groups are polypeptides of from one to about 3 amino acids, preferably of from one to about 30 amino acids.
- preferred bifunctional linkage groups can be crosslinking reagents, such as for example reagents having two reactive moieties such as succinimidyl esters, maleimides, iodoactyl groups and nitrophenyl groups
- ⁇ direct bonds Direct bonds between two protein or polypeptides are bonds that link a nitrogen, carbon, oxygen or sulfur from one protein or polypeptide to a nitrogen, carbon, oxygen or sulfur from the other protein or polypeptide.
- a fibrinogen-converting enzyme is a substance that catalyzes a conversion of fibrinogen to a derivative that spontaneously polymerizes noncovalently to form fibrin polymer.
- the derivative will be fibrin I (desAA- fibrin), fibrin II (desAA-desBB-fibrin) or desBB-fibrin.
- High affinity binding between a first substance and a second substance is binding of sufficient avidity to allow for the first or second substance to be used as an affinity ligand for the isolation of the other substance.
- high affinity binding is reflected in a association constant of about 10 ⁇ M"l or more, preferably 10° "
- Monomeric fibrin is fibrin that has been prevented from polymerization so that, for a solution form of a fibrin composition, when examined by such techniques as ultracentrifugation or gel filtration substantially all of the fibrin chain molecules in the composition behave as the non-polymerized hexamers (o-2 ⁇ 2Y2)-
- substantially all in this context means at least about 80% of the fibrin chain molecules, preferably at least about 90%, more preferably at least about 95%.
- the monomeric form in some instances will be indicated by the fact that the solid was formed from a solution form of monomeric fibrin monomer by lyophilization or another dehydration method.
- Solid forms can include suspended solids within a liquid in which the fibrin is not soluble, such as a suspension in acetone.
- ⁇ peptide or polypeptide The term "polypeptide" as used herein includes shorter polypeptides such as those often referred to a "peptides,” for instance polypeptides of less than about 100 amino acid residues.
- a recombinant protein is a protein in a form or a protein expressed in a cell in which it would not be found but for the cell or organism that expresses the recombinant protein, or an ancestor of that cell or organism, having been transformed by the introduction of extrinsic nucleic acid material.
- a snake venom-derived proteinase is a proteinase found in snake venom, a proteinase prepared from snake venom, a recombinant proteinase prepared from a cDNA for a snake venom proteinase or a portion of that cDNA which recombinant proteinase retains the proteolytic activity of the snake venom proteinase of the cDNA, a synthetically prepared snake venom proteinase or a portion of any of the foregoing that retains the proteolytic activity of the parent molecule.
- the fibrinogen-converting enzyme is preferably batroxobin ("Btx"), i.e., a proteinase from the snake venom of snakes of the genus Bothrops.
- Btx batroxobin
- Other proteinases of appropriate specificity can also be used.
- Snake venom proteinases are particularly suitable, including without limitation the venom enzymes from Agkistrodon acutus, Agkistrodon ancrod, Agkistrodon bilineatus, Agkistrodon contortrix contortrix,
- Agkistrodon halys pallas Agkistrodon (Calloselasma) rhodostoma, Bothrops asper, Bothrops atrox, Bothrops insularis, Bothrops jararaca, Bothrops Moojeni, Lachesis muta muta, Crotalus adamanteus, Crotalus atrox, Crotalus durissus terrificus, Trimeresurus flavorviridis, Trimeresurus gramineus, Trimeresurus mucrosquamatus and Bitis gabonica.
- enzymes from Bothrops are used.
- Sequences for enzymes from Trimeresurus mucrosquamatus are available from the National Center for Biotechnology Information (NCBI, Bethesda, MD) under accession numbers 602596, 602598, 602600, 602602 and 602604. Sequence for an enzyme from Protobothrops mucrosquamatus is available from the NCBI under accession number 951152. Sequence for an enzyme from Agkistrodon ancrod (Malayan pit viper, also known as Calloselasma rhodostoma) is available from the SWISS-PROT protein database (accessible through the NCBI) under accession number P47797.
- Sequence for an enzyme from Bothrops atrox is available from the NCBI under accession number 211031 or Genbank under accession number J02684. Sequence for an enzyme from Bothrops jararaca is available from the PIR protein database (accessible through the NCBI) under accession number A54361. Sequences for enzymes from Russell's viper (Vipera Russell ⁇ ) are available from the SWISS-PROT protein database (accessible through the NCBI) under accession numbers P 18964 and P 18965. Sequences for an enzyme from Trimeresurus flavoviridis is available from the SWISS-PROT protein database (accessible through the NCBI) under accession number P05620.
- Sequence for an enzyme from Crotalus atrox is available from the PIR protein database (accessible through the NCBI) under accession number A45655. Sequence for an enzyme from Agkistrodon bilineatus is available from the NCBI under accession number 211031. Sequences for enzymes from Agkistrodon contortrix are available from the NCBI under accession numbers 603215 and 603217 and from GenBank (accessible through the NCBI) under accession numbers 106680, 106681, 106724 and 106751.
- the fibrinogen-converting enzyme is thrombin
- the fusion protein of the invention will be formed by chemical methods. Recombinant methods must account for the proteolytic processing reactions required to generate thrombin from prothrombin, as was done by Falkner et al., International Patent Application WO91/11519 ("Recombinantly Produced Blood Factors").
- snake-derived proteinases that convert prothrombin to thrombin are, for example, of use in large-scaled processes for producing thrombin, in methods for producing autologous thrombin. These methods are improved by the use of enzyme preparations that can be removed through the use of a binding-partner relationship.
- the snake-derived prothrombin-converting enzyme is preferably from the venom o ⁇ Ecchis carinatus.
- Other snake venom proteinases that are suitable, include without limitation the venom enzymes from Australian tiger snake and Akistrodon hallys pallys.
- a prothrombin activator from Kenyan Echis carinatus venom is described in Kawabata, Biochemistry 34: 1771-1778, 1995 (GenBank Accession No. D32212).
- the polypeptide that is a member of a binding pair is preferably avidin or Streptavidin, which polypeptides each bind with high affinity to biotin.
- An amino acid sequence for avidin is described in Dayhoff, Atlas of Protein Sequence, Vol. 5, National Biomedical Research Foundation, Washington, DC, 1972 (see also, DeLange and Huang, J Biol. Chem. 246: 698-709, 1971), and an amino acid sequence for Streptavidin is described in Argarana et ., Nucl.
- Nucleic acid sequences are available, for example, as follows: (1) chicken mRNA for avidin, Gene Bank Ace. No. X5343, Gore et al., Nucl. AcidRes. 15: 3595-3606, 1987; (2) chicken, strain White Leghorn mRNA for avidin, Gene Bank Ace. No. L27818 (3) streptavidin from Strep, avidinii, Gene Bank Ace. No. X03591, Argarana et al., Nucl. AcidRes.
- Avidin and Streptavidin are preferably used in a tetrameric form, although monomers can be used.
- an avidin or Streptavidin protein retains biotin-binding activity, it is of course envisioned that this may involve the protein forming multimeric associations with like proteins.
- binding pair can include an antibody specific for a polypeptide or other molecule, any polypeptide to which an antibody is available or can be prepared, thioredoxin, which binds phenylarsine oxide (expression vectors include, for example, the thioredoxin fusion protein vector pTrxFus available from Invitrogen, Carlsbad, CA, or Invitrogen B.V., Netherlands), poly-His sequences that bind to divalent cations such as nickel II (expression vectors include, for example, the pThioHis vectors A, B and C available from Invitrogen), glutathione-S- transferase vectors that bind to glutathione (vector for example available from Pharmacia Biotech, Piscataway, NJ).
- expression vectors include, for example, the thioredoxin fusion protein vector pTrxFus available from Invitrogen, Carlsbad, CA, or Invitrogen B.V., Netherlands
- the binding-pairs used in the invention preferably display high affinity binding even at relatively low pH, such as a pH of about 5.
- the member of a binding pair that is attached to an enzyme is not limited to polypeptide members of binding pairs. In this case, biotin is the most preferred such member of a binding pair.
- the polypeptide chains making up the fusion proteins will be manufactured by recombinant means, as described further below. These recombinant techniques allow for the polypeptide chains to be modified by amino acid substitutions and sequence deletions such as deletions of internal or terminal sequences. Further, the N-terminal leader sequence can be modified as appropriate to promote export of the protein from the host cell. Such modified recombinant products can be readily synthesized on a small pilot scale and tested, for instance for enzymatic activity or binding activity. These pilot tests can generally be conducted without strenuous purification procedures since the organism used to produce the recombinant substance can be selected to lack the relevant activity, allowing for crude lysates or unpurified culture medium to be tested for the activity.
- Mutational and deletional approaches can be applied to all of the nucleic acid sequences encoding relevant polypeptide chains. Conservative mutations are preferred. Such conservative mutations include mutations that switch one amino acid for another within one of the following groups:
- Aromatic residues Phe, Tyr and Trp.
- a preferred listing of conservative variations is the following:
- the types of variations selected may be based on the analysis of the frequencies of amino acid variations between homologous proteins of different species developed by Schulz et al., Principles of Protein Structure, Springer- Verlag, 1978, on the analyses of structure- forming potentials developed by Chou and Fasman, Biochemistry 13, 211, 1974 and Adv. Enzymol, 47, 45-149, 1978, and on the analysis of hydrophobicity patterns in proteins developed by Eisenberg et al., Proc. Natl. Acad. Sci. USA 81, 140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157, 105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15, 321-353, 1986. All of the references of this paragraph are incorporated herein in their entirety by reference.
- the association between the fibrinogen-converting enzyme or snake-derived proteinase and the polypeptide that is a member of a binding pair is effected by recombinantly expressing the two components of the fusion proteins with (a) the two polypeptide encoding nucleic acids are directly fused such that in the synthesized protein the C-terminal amino acid of one polypeptide is directly linked by a peptide bond to the N-terminal amino acid of the other or (b) the two polypeptide encoding nucleic acids are fused via a linker nucleic acid encoding an amino acid or polypeptide, such that in the synthesized protein the C-terminal amino acid of one polypeptide is linked by a peptide bond to the N-terminal of the linker amino acid or polypeptide, which linker amino acid or polypeptide is linked by a peptide bond at its C- terminal to the N-terminal of the other polypeptide of the fusion protein.
- the fusion between the fibrinogen-converting enzyme or snake-derived proteinase and the polypeptide that is a member of a binding pair is effected by other types of bonds including disulfide bonds between cysteine residues of the respective polypeptides, amide bonds between amine and carboxylate functionalities of the two polypeptides, and bonds formed by bifunctional crosslinking reagents.
- Such bifunctional reagents include compounds with activated acyl esters such as N- hydroxysuccinimide esters, mercuric ion, other mercury compounds, compounds containing maleimide functionalities, compounds containing iodoacetyl functionalities, compounds containing fluoro-nitro-aryl functionalities, compounds containing alkylimidate functionalities, compounds containing arylsulfonyl chloride functionalities, compounds containing isocyanate functionalities, aldehyde or dialdehyde compounds and compounds containing diazoaryl functionalities.
- Crosslinking methods using such reagents are reviewed in Means and Feeney, Chemical Modification of Proteins, Holden- Day, San Francisco, 1971, which document is incorporated herein in its entirety by reference.
- N-linked glycosylations typically occur at the asparagine of Asn-Xaa-Ser/Thr tripeptide subsequences of glycoproteins. Converting Enzyme-Attachable Polypeptide Fusion Protein
- the fibrinogen converting enzyme is fused not with the member of a binding pair, but with a polypeptide designed to facilitate linkage with a member of a binding pair.
- a facilatitive polypeptide can, for instance, comprise a polylysyl polypeptide, or another repetitive polypeptide that is rich in an amino acid whose side chain is useful in linking the member of a binding pair.
- the facilitative polypeptide comprises between about 10 and about 50 amino acid residues, more preferably between about 20 and about 30 amino acid residues.
- the linkable amino acid comprises lysine, arginine, histidine, aspartic acid, glutamic acid or cysteine, more preferably lysine or cysteine, and yet more preferably lysine.
- lysine arginine
- histidine aspartic acid
- glutamic acid or cysteine more preferably lysine or cysteine
- yet more preferably lysine lysine.
- a preferred method of linking a member of a binding pair to a carbohydrate structure is oxidation with periodate followed by reductive alkylation. Recombinant Nucleic Acids.
- nucleic acid amplification methods such as polymerase chain reaction (PCR) methods, can be used to amplify useful polypeptide-encoding nucleic acids from the RNA of a tissue that expresses such a polypeptide.
- PCR polymerase chain reaction
- Such PCR methods are well described in PCR Protocols, Cold Spring Harbor Press, 1991. In some cases, PCR methods directly applied will only isolate internal sequences. Fortunately, methods have been developed to amplify and isolate sequences extended from such internal sequences so as to encompass all useful sequence.
- PCR-RACE protocols for this method are available, for example, from Gibco BRL (Gaithersburg, MD).
- amplification methods can be used to isolate two or more overlapping nucleic acids that together encode all of the needed nucleic acid, these can be pieced together using natural restriction sites or by designing restriction sites by use of appropriate PCR primers.
- restriction sites are designed into the PCR primers, it can be necessary to change the codons used to encode particular amino acid residues or to make mutational changes (preferably conservative) to design the restriction site.
- the native sequences can be used as a starting point and modified to suit particular needs.
- the sequences can be mutated to incorporate useful restriction sites. See Maniatis et al. Molecular Cloning, a Laboratory Manual (Cold Spring Harbor Press, 1989). Such restriction sites can be used to create "cassettes", or regions of nucleic acid sequence that are facilely substituted using restriction enzymes and ligation reactions.
- the cassettes can be used to substitute synthetic sequences encoding mutated enzyme or binding polypeptide amino acid sequences.
- the enzyme or binding polypeptide-encoding sequence can be substantially or fully synthetic. See, for example, Goeddel et al., Proc.
- codon usage preferences for the organism in which such a nucleic acid is to be expressed are advantageously considered in designing a synthetic enzyme or binding polypeptide-encoding nucleic acid.
- a nucleic acid sequence incorporating prokaryotic codon preferences can be designed from a mammalian-derived sequence using a software program such as Oligo-4, available from National Biosciences, Inc. (Plymouth, MN).
- the nucleic acid sequence embodiments of the invention are preferably deoxyribonucleic acid sequences, preferably double-stranded deoxyribonucleic acid sequences. However, they can also be ribonucleic acid sequences.
- the invention also relates to a mutated or deleted version of a nucleic acid sequence that encodes a protein that retains (a) the ability to bind specifically another molecule or (b) the intended enzymatic activity.
- These analogs can have N-terminal, C-terminal or internal deletions, so long as appropriate function is retained.
- a suitable expression vector is capable of fostering expression of the included polypeptide in a host cell, which can be eukaryotic (including fungal), or prokaryotic.
- Useful expression vectors include pRc/CMV (Invitrogen, San Diego, CA), pRc/RSV (Invitrogen), pcDNA3 (Invitrogen), Zap Express Vector (Stratagene Cloning Systems, LaJolla, CA); pBk/CMV or pBk-RSV vectors (Stratagene), Bluescript II SK +/- Phagemid Vectors (Stratagene), LacSwitch (Stratagene), pMAM and pMAM neo (Clontech, Palo Alto, CA), pKSVIO (Pharmacia, Piscataway, NJ), pCRscript (Stratagene) and pCR2.1 (Invitrogen), among others.
- Useful yeast expression systems include, for example, pYEUra3 (Clontech).
- Useful baculovirus vectors, for expression in insect cells include several viral vectors from Invitrogen (San Diego, CA) such as pVL1393, pVL1392, pBluBac2, pBluBacHis A, B or C, and pbacPAC ⁇ from Clontech. Some of these vectors will utilize inducible promoters such as the lac promoter.
- inducible promoters are desirable, such as promoters responsive to zinc or other metal ions, to metabolites or metabolite mimics such as isopropylthio- -galactoside, or to hormones such as estrogen or ecdyson (for instance, found in expression systems available from Invitrogen, San Diego, CA). Inducible systems help to minimize the adverse effects that can flow from the expressed protein having toxic effects on the expression cells.
- the polypeptides are preferably expressed in a mammalian cell line, preferably a transformed cell line with an established cell culture history.
- suitable cell lines include COS-1, COS-7, LM(tk"), HeLa, HEK293, CHO, Rat-1 and NIH3T3.
- the polypeptides are expressed in a cell line that is more inexpensively maintained and grown than are mammalian cell lines, such as a bacterial cell line or a fungal cell line such as a yeast cell line.
- mammalian cell lines such as a bacterial cell line or a fungal cell line such as a yeast cell line.
- E. coli bacterial cells are particularly preferred.
- One simplified method of isolating polypeptides synthesized by an organism under the direction of one of the nucleic acids of the invention is to recombinantly express a version of the fusion protein having a fusion component that is facilely affinity purified.
- This fusion component can be simply the polypeptide chain that is a member of a binding pair. Or, this fusion component can be another fused polypeptide.
- a useful component for purification is, for instance, glutathione S-transferase, which is encoded on commercial expression vectors (e.g., vector pGEX4T3, available from Pharmacia, Piscataway, NJ).
- Another useful purification component is, for instance, thioredoxin.
- This glutathione S-transferase-containing fusion protein can then be purified on a glutathione affinity column (for instance, that available from Pharmacia, Piscataway, New Jersey). If extra fusion partners are used, the extra fusion partner can be removed by partial proteolytic digestion approaches that preferentially attack unstructured regions such as the linkers between the extra fusion partner and the desired fusion protein.
- the linkers can be designed to lack structure, for instance using the rules for secondary structure forming potential developed, for instance, by Chou and Fasman, Biochemistry 13, 211, 1974 and Chou and Fasman, Adv. in Enzymol. 47, 45-147, 1978.
- the linker can also be designed to incorporate protease target amino acids, such as, arginine and lysine residues, the amino acids that define the sites cleaved by trypsin.
- protease target amino acids such as, arginine and lysine residues
- standard synthetic approaches for making oligonucleotides can be employed together with standard subcloning methodologies.
- Other fusion partners besides GST can be used. Procedures that utilize eukaryotic cells, particularly mammalian cells, are preferred since these cells will post-translationally modify the protein to create molecules highly similar to or identical to native proteins.
- the protein is substantially pure, meaning a purity of at least 20%> w/w with respect to other proteins, more preferably at least about 50%>, yet more preferably at least about 70%>, still more preferably at least about 90%>.
- the fusion protein is "isolated” if it has been separated from other proteins or other macromolecules of the cell or tissue from which it is derived or prepared.
- blood is for example drawn from a patient and mixed with an anticoagulant, such as trisodium citrate to a final concentration of about 0.5% w/v.
- an anticoagulant such as trisodium citrate
- Plasma is isolated by centrifugation, which removes the cellular components of the blood.
- the fusion protein is added to the fibrinogen containing solution, for example, at a concentration approximately corresponding, on a molar basis, to a concentration of baxtroxobin of about 0.1 ⁇ g/ml to about 100 ⁇ g/ml, preferably to a concentration of about 0.5 ⁇ g/ml to about 50 ⁇ g/ml.
- a precipitate of fibrin polymer forms from the reaction of fibrinogen with enzyme incorporated into the fusion protein.
- fibrinogen-converting enzyme is batroxobin
- the polymer is generally made up of fibrin I.
- the fibrin polymer is isolated by centrifugation or filtration, and then dissolved in a low pH buffer, such as 0.2 M sodium acetate, pH 4.0, preferably in the presence of calcium ions, for example at a concentration of about 20 mM.
- a low pH buffer such as 0.2 M sodium acetate, pH 4.0
- the biotinylated support can be prepared, for example, by reacting one of the biotinylating agents available from Boehringer Manheim (Indianapolis, IN) or Clontech (Palo Alto, CA) with a solid support having primary amino groups.
- the biotinylating reagents typically have a biotinyl substituent, one to two aminocaproyl spacer groups, and a reactive N-hydroxysuccinimide group.
- the solid support can be, for example, the amino-derivatized agarose resins available from Sigma (St. Louis, MO) or an amino-derivatized chromatography matrix available from Pharmacia (Uppsala, Sweden).
- the solubilized fibrin is removed from the support-bound fusion protein by centrifugation or filtration.
- the solubilized fibrin is now ready for use in a sealant, for instance as described in Edwardson et al., European Patent Application No. 592,242.
- Preparation of Solid Supports The solid support to which the second member of the binding pair is bound is, for example made up of beaded or non-beaded particles of carbohydrate-based material such as agarose, cross-linked agarose or cross-linked dextran, or a non-porous material such as polystyrene.
- Methods for covalently coupling molecules to solid supports include for example creating reactive sites on the solid supports with cyanogen bromide or reacting the solid supports with bifunctional reagents such as diglycidyl ethers. See, for example, "Attachment to Solid Supports” in Means and Feeney, Chemical Modification of Proteins, Holden-Day, San Francisco, 1971, pp. 40-43 ox Affinity Chromatography: A Practical Approach, Dean et al., eds., IRL Press, Oxford, 1991, the disclosures of which two references are incorporated herein in there entirety by reference.
- alkyloxysilane moieties for example, can provide the silica-reactive moiety of a bifunctional coupling reagent.
- ⁇ -glycidoxypropyltrimethoxysilane can be reacted with the silica-based material, which is then directly reacted with the protein (via the glycidic ether moiety), or a second step is employed such as reacting the glycidic ether with an amine and subsequently attaching by reductive alkylation a glycoprotein that is mildly oxidized (for instance with periodate) to contain aldehyde moieties.
- a preferred coupling chemistry reacts a carbohydrate-based solid support with a hydrazide group, and then coupling by reductive alkylation a glycoprotein that is mildly oxidized (for instance with periodate) to contain aldehyde moieties. See, Axelsson et al., Thromb. Haemost. 36: 517, 1976, which document is incorporated herein in its entirety by reference.
- a EcoRT-Xbal fragment encoding batroxobin is excised from a pUC18 clone (R&D Systems, Inc., Minneapolis, MN) and cloned into the multiple cloning site of pCI- neo (Promega, U.K., Southampton, UK).
- the expression sequence is made up of the -24 to 228 sequence of batroxobin which includes the leader sequence.
- the batroxobin enzyme is expressed in CHO cells from the resulting vector.
- Example IB Bacterial Vector Encoding Batroxobin-thioreductase fusion Protein
- a Bsal-Xbal fragment encoding batroxobin was excised from a pCI/neo clone (R&D Systems, Inc., Minneapolis, MN) and cloned into the multiple cloning site of pTrxFus (Invitrogen).
- the resulting fusion protein expressed in E. coli was made up of thioredoxin fused at its 5' end via linker with an enterokinase cleavage site to the 1 to 228 amino acid sequence of batroxobin.
- the fusion protein was purified phenylarsine oxide column (Invitrogen, B.V., Netherlands).
- Example 2 Chemical Formation of a Batroxobin- Avidin Fusion Protein Covalent complexes may be formed between batroxobin and avidin using
- N-succinimidy-3-(2-pyridyldithio)proprionate (SPDP, Pierce Chemical Co., Rockford, IL) as follows: 3mg of batroxobin was reacted with 1.9 mg SPDP dissolved in 0.75 ml ethanol for 60 minutes at room temperature. 6 mg avidin was reacted identically in a separate container. The protein product of each reaction was separately desalted on Sephadex G25 in 50 mM sodium phosphate, 20 mM NaCl, pH 7.0 buffer. The derivatised proteins were then activated by reduction, which exposed thiol groups derived from the SPDP, mixed together, and the mixed proteins were again desalted by gel filtration on Sephadex G25. Batroxobin-avidin conjugates were isolated (away from non- conjugated protein) by gel filtration on Sephadex G100.
- SEQ ID NO: 1 is of the Gallus gallus cDNA for avidin, and SEQ ID NO: 2 is the corresponding protein.
- Amino acids 1-24 are believed to be the leader sequence, and amino acids 25-152 the mature protein.
- SEQ ID NO:3 is an cDNA for streptavidin, and SEQ ID NO:4 is the corresponding protein.
- Amino acids 1-24 are believed to be the leader sequence, and amino acids 25-183 the mature protein.
- SEQ ID NO:5 is of the Bothrops atrox cDNA for batroxobin, and SEQ ID NO: 6 is the corresponding protein.
- Amino acids 1-18 are believed to be the leader sequence, and amino acids 25-255 the mature protein.
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Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2000524411A JP2001526027A (en) | 1997-12-09 | 1998-12-09 | Fibrinogen converting enzyme hybrid |
CA002312476A CA2312476A1 (en) | 1997-12-09 | 1998-12-09 | Fibrinogen-converting enzyme hybrids |
AU19058/99A AU753458B2 (en) | 1997-12-09 | 1998-12-09 | Fibrinogen-converting enzyme hybrids |
EP98963813A EP1037969A4 (en) | 1997-12-09 | 1998-12-09 | Fibrinogen-converting enzyme hybrids |
NO20002937A NO20002937L (en) | 1997-12-09 | 2000-06-08 | Fibrinogen converting enzyme hybrids |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US6797897P | 1997-12-09 | 1997-12-09 | |
US60/067,978 | 1997-12-09 |
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WO1999029838A1 true WO1999029838A1 (en) | 1999-06-17 |
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Application Number | Title | Priority Date | Filing Date |
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PCT/US1998/026086 WO1999029838A1 (en) | 1997-12-09 | 1998-12-09 | Fibrinogen-converting enzyme hybrids |
Country Status (6)
Country | Link |
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EP (1) | EP1037969A4 (en) |
JP (1) | JP2001526027A (en) |
AU (1) | AU753458B2 (en) |
CA (1) | CA2312476A1 (en) |
NO (1) | NO20002937L (en) |
WO (1) | WO1999029838A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
SG127720A1 (en) * | 1999-10-01 | 2006-12-29 | Amgen Inc | Fibrinolytically active polypeptide |
US7993893B2 (en) * | 2006-10-19 | 2011-08-09 | Konruns Pharmaceutical Co., Ltd | Haemocoagulase |
DE102014112212A1 (en) * | 2014-08-26 | 2016-03-03 | Akesion Gmbh | Recombinant fusion proteins for the prevention or treatment of adhesions in tissues or organs |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997017436A1 (en) * | 1995-11-09 | 1997-05-15 | Bristol-Myers Squibb Company | Stable avidin composition and methods using same |
WO1997017435A1 (en) * | 1995-11-09 | 1997-05-15 | Bristol-Myers Squibb Company | Stable biotinylated biomolecule composition and methods |
Family Cites Families (1)
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CN1091315A (en) * | 1992-10-08 | 1994-08-31 | E·R·斯奎布父子公司 | Fibrin sealant compositions and using method thereof |
-
1998
- 1998-12-09 EP EP98963813A patent/EP1037969A4/en not_active Withdrawn
- 1998-12-09 AU AU19058/99A patent/AU753458B2/en not_active Ceased
- 1998-12-09 CA CA002312476A patent/CA2312476A1/en not_active Abandoned
- 1998-12-09 WO PCT/US1998/026086 patent/WO1999029838A1/en not_active Application Discontinuation
- 1998-12-09 JP JP2000524411A patent/JP2001526027A/en active Pending
-
2000
- 2000-06-08 NO NO20002937A patent/NO20002937L/en not_active Application Discontinuation
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997017436A1 (en) * | 1995-11-09 | 1997-05-15 | Bristol-Myers Squibb Company | Stable avidin composition and methods using same |
WO1997017435A1 (en) * | 1995-11-09 | 1997-05-15 | Bristol-Myers Squibb Company | Stable biotinylated biomolecule composition and methods |
US5691152A (en) * | 1995-11-09 | 1997-11-25 | E. R. Squibb & Sons, Inc. | Stable avidin composition |
Non-Patent Citations (12)
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
SG127720A1 (en) * | 1999-10-01 | 2006-12-29 | Amgen Inc | Fibrinolytically active polypeptide |
US7993893B2 (en) * | 2006-10-19 | 2011-08-09 | Konruns Pharmaceutical Co., Ltd | Haemocoagulase |
DE102014112212A1 (en) * | 2014-08-26 | 2016-03-03 | Akesion Gmbh | Recombinant fusion proteins for the prevention or treatment of adhesions in tissues or organs |
WO2016030278A1 (en) * | 2014-08-26 | 2016-03-03 | Akesion Gmbh | Recombinant fusion proteins for preventing or treating adhesions of tissues or organs |
US10837007B2 (en) | 2014-08-26 | 2020-11-17 | Akesion Gmbh | Recombinant fusion proteins for preventing or treating adhesions of tissues or organs |
Also Published As
Publication number | Publication date |
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AU753458B2 (en) | 2002-10-17 |
AU1905899A (en) | 1999-06-28 |
NO20002937L (en) | 2000-08-08 |
JP2001526027A (en) | 2001-12-18 |
NO20002937D0 (en) | 2000-06-08 |
CA2312476A1 (en) | 1999-06-17 |
EP1037969A1 (en) | 2000-09-27 |
EP1037969A4 (en) | 2003-05-21 |
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