JP2001526027A - Fibrinogen converting enzyme hybrid - Google Patents

Abstract

  (57) [Summary] A fusion 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 Can directly (1) utilize an N-terminal amino group, a C-terminal carboxy group or a side chain functional group to the first polypeptide chain, or (2) provide a divalent linking component for linking such a group or functional group. To provide a fusion protein that is linked to the fusion protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a multidomain protein comprising a first polypeptide which is a fibrinogen converting enzyme or a proteinase derived from snake toxin and a second polypeptide which is a member of a binding pair, wherein the other component of the binding pair is Members can be used to remove the fusion protein from the fibrin preparation formed through the action of this converting enzyme. The present invention further relates to a recombinant method for forming the multidomain protein, a nucleic acid and a vector used in the method, and a method for forming fibrin using the fusion protein. The invention further provides a fusion protein of a fibrin converting enzyme and a polypeptide designed to promote the covalent binding of one member of a binding pair. The present invention also provides an aggregate of (a) a first polypeptide covalently linked to a member of a binding pair and (b) a second polypeptide that first binds to the member of the binding pair by binding to the member. I do.

[0002] "Fibrin" sealants are widely used to control bleeding in surgery and to stop incised blood vessels and tissues in either surgery or wounds. "Fibrin" is interpreted in this context as a misnomer, since historically "fibrin" hemostats have been introduced as a precursor to fibrin, a substance containing fibrinogen. In such hemostatic agents, the fibrinogen substance has been introduced into the site to be sealed, along with a proteinase enzyme that converts fibrinogen to fibrin. When a sufficient amount of fibrin is formed from fibrinogen, fibrin polymerizes spontaneously into a fibrin polymer, and when sufficient polymer is collected, a fibrin clot is formed. Generally, the converting enzyme has been bovine thrombin. Recently, however, effective haemostatic agents have been described that carry fibrin to the site where hemostasis is to be carried out with polymerization inhibited. At this site, the polymerization inhibition condition is reversed and an effective clot is formed.
See Edwardson et al., European Patent Application EP 592,242. EP59
As described in 2,242, fibrin in a hemostatic agent can be formed by contacting fibrinogen with a fibrinogen converting enzyme bound to a solid support. This solid support allows removal of the converting enzyme from the hemostat.

[0003] One of the particular advantages of the fibrin hemostatic agent of EP 592,242 is that it can be an auto-hemostatic agent which is immediately prepared from a small amount of patient's blood in just one minute before surgery, and This preparation can be performed with standard hospital equipment. Prior art methods for deriving the fibrinogen material of hemostatic agents are much more demanding and difficult to automate. Specialized procedures for preparing fibrin have also recently been described, and such procedures are rapidly, highly reproducible, highly reliable, and highly safe to remove autologous hemostats from patients. To be prepared. Holm `` Centrifuge Reagent Delivery System '' WO 96/16713, Holm
`` Method and Device for Separating Fibrin I from Blood Plasma '' WO 96 /
See 16714 and Holm "Centrifuge with Annular Filter" WO 96/16715.

[0004] The present invention provides an additional means for removing fibrinogen converting enzyme from a fibrin hemostat preparation. In one aspect, the invention relates to a fusion protein, which comprises the convertase and another convertible enzyme that can be used to bind the fusion protein to a solid support after it has been used to form fibrin from fibrinogen. And a fusion protein comprising: In another aspect, the invention provides a fusion protein comprising the converting enzyme and another polypeptide that can be used to covalently bind a binding component. In yet another perspective,
The convertase and other polypeptides are aggregated via the other polypeptide bound to a member of the binding pair covalently linked to the convertase.

[0005] In a first embodiment Summary of the Invention, the present invention is a multidomain protein, a second polypeptide comprising a first member of a first polypeptide chain and binding pair comprising fibrinogen converting enzyme Wherein the second polypeptide chain is directly attached to the first polypeptide chain by (1) an N-terminal amino group, a C-terminal carboxy group or a linkage utilizing a side chain functional group; Linked via a divalent linking moiety linking these groups or functional groups, or (3) by a first member linked to a second member of the binding pair covalently linked to the first polypeptide chain. Yes,
Provide multi-domain proteins. In one embodiment, the multidomain protein is a recombinant protein comprising a contiguous amino acid sequence comprising a second polypeptide chain and a first polypeptide chain, and the second polypeptide chain is a polyprotein having biotin binding activity. Comprising a peptide. The second polypeptide chain may comprise a multivalent conjugate, eg, an antibody having more than one binding site, streptavidin or avidin. Such a multivalent conjugate can bind to the first polypeptide via a ligand at a binding site covalently attached thereto, leaving at least one other binding site effective for binding another ligand molecule.

[0006] In a second aspect, the invention includes a contiguous amino acid sequence comprising 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 fused directly via peptide bonds or fused via a linker polypeptide chain.

[0007] In a third aspect, the present invention provides a first polypeptide comprising fibrinogen converting enzyme and an amino acid side chain or (b) a binding partner that can be used to add two or more (a) binding partners. O-linked or N available to add
-A recombinant fusion protein comprising a continuous polypeptide chain comprising a second polypeptide comprising a linking polysaccharide. In one embodiment, the amino acid residues of the second polypeptide are selected to minimize the degree of secondary structure formed near the amino acid having an appendable side chain.

[0008] In a fourth aspect, the invention is a method of preparing a fibrin composition, comprising:
1) providing a method comprising forming a fibrin composition by contacting a composition comprising fibrinogen with an enzyme effective to convert fibrinogen to fibrin, wherein the enzyme comprises a fibrinogen converting enzyme And a second polypeptide chain comprising a first polypeptide chain comprising a first member of a binding pair, wherein the second polypeptide chain comprises the first polypeptide chain. (A) directly to the peptide chain by a bond utilizing an N-terminal amino group, a C-terminal carboxy group or a side chain functional group, (b) via a divalent linking component connecting these groups or functional groups, or (c) ) Linked by a first member bound to a second member of a binding pair that is covalently bound to the first polypeptide chain.
Preferably, when the fibrin composition is not a monomeric fibrin composition,
The method further comprises the step of (2) forming a monomeric fibrin composition from the fibrin composition. Preferably, the method further comprises (3) contacting the monomeric fibrin composition with a solid support to which the second member of a binding pair effective to bind the first member is bound. In another preferred aspect, the method comprises, after step 3), (4) removing the solid support and recovering the monomeric fibrin composition thus obtained.

[0009] In a fifth aspect, the present invention provides an alpha polypeptide chain comprising a snake venom derived proteinase effective to convert prothrombin to thrombin;
A conjugation protein comprising a second molecule that is a member of a binding pair covalently linked to the alpha polypeptide chain.

[0010] In a sixth aspect, the present invention provides an alpha polypeptide chain comprising a snake venom derived proteinase effective to convert prothrombin to thrombin;
A nucleic acid encoding a recombinant fusion protein comprising a contiguous amino acid sequence comprising a beta polypeptide chain comprising members of a binding pair, wherein the alpha and beta polypeptides are linked via peptide bonds. Directly or fused via a linker polypeptide chain.

[0011] In a seventh aspect, the invention provides a method of preparing a thrombin composition, comprising:
1) contacting a composition comprising prothrombin with a snake venom enzyme effective to convert prothrombin to thrombin, thus forming a thrombin composition, wherein the enzyme comprises the enzyme Comprising a multidomain protein comprising an alpha polypeptide chain and a beta polypeptide chain comprising the first member of the binding pair, wherein the beta polypeptide chain comprises (a )) Directly through a linkage utilizing an N-terminal amino group, a C-terminal carboxy group or a side chain functional group, (b) via a divalent linking component linking these groups or functional groups, or (c) a Linked by a first member that binds the two members, wherein the second member of the binding pair is the first polypeptide. It is covalently bonded to the chain. Preferably, the method further comprises the step of (2) contacting the thrombin composition with the bound solid support of the second member of the binding pair effective to bind the first member. Become. In another preferred aspect, the method further comprises (3) removing the solid support and recovering the thrombin composition thus obtained.

Definitions -Alpha, beta, first, second; terms such as "first,""second,""alpha," and "beta" are used herein to help distinguish between descriptions of similar elements. Use it as a generic name.

Divalent linking group: A divalent linking group refers to a molecule having two sites for adding a protein or polypeptide. Preferred divalent linking groups are polypeptides of 1 to 3 amino acids, preferably 1 to about 30 amino acids. Further, suitable divalent linking groups are crosslinkers, such as two reactive components, such as succinimidyl esters, maleimides,
It is a reagent having an iodoactyl group and a nitrophenyl group.

[0014] Direct bond: A direct bond between two proteins or polypeptides is a direct bond between nitrogen, carbon, oxygen or sulfur from one protein or polypeptide and nitrogen, carbon, oxygen or sulfur from another protein or polypeptide. A bond linked to sulfur.

Fibrinogen converting enzyme: Fibrinogen converting enzyme refers to a substance that catalyzes the conversion of fibrinogen to a derivative that spontaneously non-covalently polymerizes to form a fibrin polymer. Generally, this derivative is a fibrin I (desAA-
Fibrin), fibrin II (desAA-desBB-fibrin), or d
esBB-fibrin.

High-affinity binding: High-affinity binding between a first substance and a second substance means that the first or second substance is used as an affinity ligand for the isolation of the other substance A greedy bond that is enough to make it possible. Typically, high affinity binding is reflected by a binding constant of about 10 ⁵ M ⁻¹ or more, preferably 10 ⁶ M ⁻¹ or more, more preferably 10 ⁷ M ⁻¹ or more.

Monomeric fibrin: monomeric fibrin refers to fibrin that has been inhibited from polymerizing, and thus, in solution form of the fibrin composition, when examined by techniques such as ultracentrifugation or gel filtration, in this composition In addition, all fibrin chain molecules behave as non-polymerized hexamers (α ₂ β ₂ γ ₂ ). "Substantially all" in this context means at least about 80%, preferably at least about 90%, of the fibrin chain molecules,
More preferably, it means at least about 95%. The monomer may sometimes be shown in a solid form, since the solid may be formed from the monomeric fibrin monomer solution form by lyophilization or other dehydration methods.
If necessary, more complex analytical techniques may be applied to ensure that the fibrin hexamer does not aggregate by polymer-forming non-covalent bonds of the type and strength involved in the formation of fibrin clots. Solid forms may include suspended solids in a liquid in which fibrin is not soluble, such as an acetone suspension.

Peptide or polypeptide: As used herein, the term “polypeptide” refers to shorter polypeptides, such as those often referred to as “peptides”, eg, less than about 100 amino acid residues. Group of polypeptides.

Recombinant protein: A recombinant protein is a protein and refers to a form of a protein that is expressed only in cells or organisms transformed by introduction of an exogenous nucleic acid substance, or progeny of the cells or organisms. .

Snake toxin-derived proteinase; snake venom-derived proteinase is a proteinase found in snake venom, a proteinase prepared from snake venom, a recombinant proteinase prepared from snake venom proteinase cDNA, or a part of the cDNA. Wherein the recombinant proteinase retains the proteolytic activity of the snake venom proteinase of the cDNA, a snake venom proteinase prepared synthetically or any of the above that retains the proteolytic activity of the parent molecule, It is.

DETAILED DESCRIPTION The enzyme-binding polypeptide fusion protein fibrinogen converting enzyme is preferably batroxobin ("Btx"), a proteinase derived from the snake venom of the Boslopus snake. Snake venom proteinases such as, but not limited to, Agkistrodon actus, Agkistrodone ancrod, Agkistrodone virineatus (A. bilin)
eatus), Agkistrodon Contortricus Contortrix, Agkistrodon Harris Paras, Agkistrodon (Carotherasum) Rhodostoma, Boslops Aspar, Boslops Atrox, Boslops Insularis, Boslops Jalalaka, Boslops Mojeta, Lacemta Sulta Crostemus Snake venoms from Adamanteus, C. atrox, C. aurex, C. aurex, C. aurex, C. aurex, C. atrox, C. atrox, C. atrox are very suitable.
In one embodiment, a Boslopus-derived enzyme is used.

[0022] The sequence of a fibrinogen converting enzyme from A. rhodostoma is described in Bach et al., WO 90/06362. Agkistrodon C.I. Two sequences from Contortrix are described in Valenzuela et al., EP03.
No. 23722. The sequence of batroxobin (derived from Boslopus atrocus mugeni) is described in Japanese Patent Application Laid-Open Publication No. H2-124092. The sequence of batroxobin, an enzyme derived from Trimeresus flavobiridis, and the sequence of the enzyme derived from Crotalus horridus are Pirk.
le and Theodor `` Structure of Thrombin-like snake venom Proteinases '' Medi
cal use of Snake Venom Proteins. Agkistrodon C.I. The sequences of Contorrix, Russell venomous snake, Bostrops atroxys mugeni and Trimelesurus flavoviridis are disclosed by McMullen et al., Biochemi.
stry 28: 674-679, 1989.

[0023] The sequence of the enzyme from Trimellesus mucloscamatus is described in the National Center of the
Accession number 602596 from Biotechnology Information (NCBI, Bethesda, MD)
, 602598, 602600, 602602 and 602604. Protobothrops mucrosquamatus (Protobothrops mucrosquamatus) (
The sequence of the enzyme from the Malai venom snake (also known as Calloselasma rhodostoma) is available from the SWISS-PROT protein database (accessible via NCBI) under accession number P47797. The sequence of the enzyme from Boslopus atrox can be obtained from NCBI under accession number 211031 or Genb.
Available from ank under accession number J02684. The sequence of the enzyme from Boslops jalaraca can be obtained from the PIR protein database (accessible via NCBI) under accession number A54361. Russell snake venom (Bipella Lasselli [
Vipera Russelli]) is derived from the SWISS-PROT protein database (accessible via NCBI) under accession numbers P18964 and P18.
965. The sequence of the enzyme derived from Trimeresus flavobiridis is S
It is available from the WISS-PROT protein database (accessible via NCBI) under accession number P05620. The sequence of the enzyme from Crotalus atrox is available under the accession number A45655 from the PIR protein database (accessible via NCBI). The sequence of the enzyme from Agkistrodon virneatus can be obtained from NCBI under accession number 211031. Agkistrodon Contortrix-derived enzyme was obtained from NCBI under accession numbers 603215 and 603217 and Ge
Accession numbers I06680, I06681 from nBank (accessible via NCBI)
, I06724 and I06751.

When the fibrinogen converting enzyme is thrombin, the fusion proteins of the invention will generally be formed by chemical methods. The recombination method is utilized for the proteolytic processing reactions required to produce thrombin from prothrombin, as practiced by Falkner et al., International Patent Application WO 91/11519 ("Recombinantly Produced Blood Factors"). There must be.

[0025] Snake-derived proteinases that convert prothrombin to thrombin are useful, for example, in large scale processes for producing thrombin in methods for producing autologous thrombin. These methods are improved by the use of enzyme preparations that can be removed via the use of binding partner relationships. The snake-derived prothrombin converting enzyme is preferably derived from the snake venom of Ecchis carinatus. Other snake venom proteinases that are suitable include, without limitation,
Snake venom enzymes from Australian Tiger Snake and Axtrodon Harris Paris. For example, the sequence of a prothrombin activator (ecarin) derived from Kenyan Echis carinatus snake venom is described in Kawabata, Biochemistry 34: 1771-1778, 1995 (GenBank accession number D32212).

The polypeptides that are members of the binding pair are preferably avidin or streptavidin, each of which binds with high affinity to biotin. The amino acid sequence of avidin is described in Dayhoff, Atlas of Protein Sequence, Vol. 5,
National Biomedical Research Foundation, Washington, DC, 1972 (also De
Lange and Huang, J. Biol. Chem. 246: 698-709, 1971), and the amino acid sequence of streptavidin is described by Argarana et al. Nucl. Acid Res. 14: 1871-18.
82, 1986. For example, nucleic acid sequences are available as follows: (1)
Chicken avidin mRNA, Gene Bank Acc. No. X5343, Gore et al. Nucl. Acid Re
s. 15: 3595-3606, 1987; (2) mRN of avidin of chicken White Leghorn strain
A, Gene Bank Acc. No. L27818 (3) Streptavidin from Strep. Avidinii, Gene Bank Acc. No. X03591, Argarana et al. Nucl. Acid. Res. 14: 1871-1882, 19
86; (4) Strep. Avidinii-derived synthetic gene for streptavidin, Gene Ban
k Acc. No. A00743, Edwards, WO 89/03422; and (5) Synthetic gene for streptavidin Gene Bank Acc. No. X65082, Thompson et al. Gene 136: 243-246, 1993.

Avidin and streptavidin are preferably used in a tetramer, but monomers can also be used. The description herein that an avidin or streptavidin protein retains biotin binding activity can, of course, be understood to include proteins that form large associations with similar proteins. Other useful members of the binding pair include antibodies specific for the polypeptide or other molecule, any polypeptide for which antibodies can be obtained or made, thioredoxin (
It binds to phenylarsine oxide (expression vectors include, for example, Invitr
a thioredoxin fusion protein vector pTrxFus available from ogen, Carlsbad, CA or Invitrogen BV, Netherlands), a poly-His sequence that binds to a divalent cation such as nickel II (expression vectors include, for example, Invitr
pThioHis vectors A, B and C available from ogen) and glutathione-S-transferase vectors that bind to glutathione (for example, vectors available from Pharmacia Biotech, Piscataway, NJ). Methods for producing such antibodies will be understood by those skilled in the art based on the polypeptide expression systems described herein and known antibody production methods. For methods of producing antibodies, for example, Au
subel et al., Short Protocols in Molecular Biology, John Wiley & Sons, New Y
ork, 1992. The binding pairs utilized in the present invention preferably exhibit high affinity binding even at relatively low pH, eg, at a pH of about 5. In some aspects of the invention, the members of the binding pair that are added to the enzyme are not members of a polypeptide binding pair. In this case, biotin is the most preferred such binding pair member.

In many embodiments of the invention, the polypeptide chains that make up the fusion protein will be produced by recombinant means, as described in more detail below. Such recombination techniques allow for modification of the polypeptide chain by amino acid substitutions and sequence deletions, such as deletion of internal or terminal sequences. Furthermore, the N-terminal leader sequence can be appropriately modified to facilitate the transport of proteins from host cells. Such modified recombinant products can be readily synthesized on a small pilot scale, and can be tested for, for example, enzymatic or binding activity. Such pilot tests can generally be performed without extensive purification procedures, because the organisms used to make the recombinant material can be selected to lack the relevant activity, crude lysate or impure culture media. This makes it possible to test the activity of the compound.

The mutation and deletion approach is applicable to all nucleic acid sequences encoding the relevant polypeptide chains. Conservative mutations are preferred. Such conservative mutations include switching one amino acid to another amino acid within the following groups: Small fatty nonpolar or slightly polar residues: Ala, Ser, Thr, Pr
o and Gly; 2. Polar negatively charged residues and their amides: Asp, Asn, Glu and Gln; 3. Polar positively charged residues: His, Arg and Lys; Large fatty non-polar residues: Met, Leu, Ile, Val and Cys;
And 5. Aromatic residues: Phe, Tyr and Trp.

A preferred list of conservative mutations is as follows: original residue changes Ala Gly, Ser Arg Lys Asn GIn, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala, Pro His Asle, Gln Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Tyr, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe ValI

The types of mutations of choice are described in Schulz et al., Principles of Protein Structure, Springe
Analysis of the frequency of amino acid mutations between homologous proteins of various species developed by r-Verlag, 1978, Chou and Fasman, Biochemistry 13, 211, 1974 and Adv. Enzymo
1, 47, 45-149, 1978.
Proc. Natl. Acad. Sci. USA 81, 140-144, 1984; Kyte &Doolittle; J. Molec.B
iol. 157, 105-132, 1981 and Goldman et al., Ann. Rev. Biophys. Chem. 15, 321-35.
3, 1986, based on analysis of hydrophobic patterns in proteins. The references in this paragraph are all incorporated herein by reference.

In a preferred embodiment of the invention, the association between the fibrinogen converting enzyme or snake-derived proteinase and the polypeptide that is a member of the binding pair comprises: (a) two components of the fusion protein The peptide-encoding nucleic acid is directly fused, such that the C-terminal amino acid of one polypeptide is directly linked to the other N-terminal amino acid by a peptide bond in the synthetic protein; or (b) the two polypeptide-encoding nucleic acids are amino acids or Fused via a linker nucleic acid encoding a polypeptide, wherein the C-terminal amino acid of one polypeptide in the synthetic protein is linked to the N-terminus of the linker amino acid or polypeptide by a peptide bond;
It is further performed by recombinant expression such that the linker amino acid or polypeptide is linked at its C-terminus to the N-terminus of the other polypeptide of the fusion protein by a peptide bond.

In other embodiments, the fusion between the fibrinogen converting enzyme or snake-derived proteinase and a polypeptide that is a member of a binding pair comprises a disulfide bond between cysteine residues of the corresponding polypeptide, two disulfide bonds. This may be achieved by other types of bonds, such as amide bonds between the amine and carboxylic acid functions of the polypeptide, and bonds formed by divalent crosslinkers. Such divalent reagents include compounds having activated acyl esters, such as N-hydroxysuccinimide esters, mercury ions, other mercury compounds, compounds containing a maleimide functional group, compounds containing an iodoacetyl functional group, fluoro- Compounds containing a nitro-aryl function, compounds containing an alkylimidate function, compounds containing an arylsulfonyl chloride function, compounds containing an isocyanate function, aldehyde or dialdehyde compounds, and compounds containing a diazoaryl function include Can be A crosslinking method using such a reagent is described in Means and Feeney, Chemical Modification of Proteins, Holden-
Day, San Francisco, 1971, the contents of which are incorporated herein by reference.

Among the modifications that can be made to proteins by means of recombinant methods, the sequences are glycosylated, especially when the protein is expressed in suitable cells. N-linked glycosylation generally occurs at asparagine in the Asn-Xaa-Ser / Thr tripeptide subsequence of the glycoprotein.

[0035] In one embodiment of the converting enzyme addable polypeptide fusion proteins present invention, the fibrinogen-converting enzyme is not fused to the member of the binding pair, designed to facilitate the connection between members of binding pairs Fused to the polypeptide. Such facilitating polypeptides include, for example,
Includes polylysyl polypeptides or other repetitive polypeptides whose side chains are rich in amino acids useful for linking members of a binding pair. Preferably, the facilitating polypeptide is about 10 to about 50 amino acid residues, more preferably about 20 amino acid residues.
Comprising up to about 30 amino acid residues. Preferably, the linkable amino acids include lysine, arginine, histidine, aspartic acid, glutamic acid or cysteine, more preferably lysine or cysteine, and even more preferably lysine. Methods for linking biotin to facilitating polypeptides are described, for example, in Savage et al., Avidin. Biotin Chemistry: A Handbook, Pierce Chemical.
Co., 1992. A preferred method of linking the members of a binding pair to a carbohydrate structure is oxidation with periodate followed by reductive alkylation.

Recombinant nucleic acids, cells and methods If the nucleic acid sequence is known, or if the amino acid sequence is sufficient to deduce a useful primer, nucleic acid amplification methods such as the polymerase chain reaction (
PCR) methods can be used to amplify such polypeptide-encoding nucleic acids from RNA in tissues that express useful polypeptides. Such a PCR method is described in PCR Protocols, Col.
d Spring Harbor Press, 1991. In some cases, the PCR method is applied directly to isolated internal sequences only. Preferably, methods have been developed to amplify and isolate sequences starting from such internal sequences to encompass all useful sequences. One such method is called PCR-RACE, and a protocol for this method is available, for example, from GIBCO BRL (Gaithersburg, MD). If amplification methods are available to isolate two or more overlapping nucleic acids that together encode all of the required nucleic acids, they may be restricted by utilizing natural restriction sites or by using appropriate PCR primers. By designing the parts, they can be cut off together. When designing a restriction site into a PCR primer, it is necessary to change the codon used to encode a particular amino acid residue or make a mutation (preferably conservative) to design the restriction site Sometimes. In some cases, it may be necessary to subclone specific nucleic acid fragments and manipulate the necessary restriction sites using well-established mutagenesis techniques. Ausubel et al., Curren
t Protocols in Molecular Biology, John Wiley and Sons, New York, 1995, p
p.8.1.1-8.1.6.

To construct a nucleic acid encoding a non-naturally occurring enzyme or binding polypeptide, the native sequence may be used as a starting point and modified to meet specific needs. For example, the sequence can be mutated to incorporate a useful restriction site. Maniatis et al.
Molecular Cloning, a Laboratory Manual (Cold Spring Harbor Press, 1989)
checking ... Such restriction sites can be used to construct "cassettes" or nucleic acid sequence regions that are easily replaced using restriction enzymes and ligation reactions. Such a cassette can be used to replace a synthetic sequence encoding the amino acid sequence of a mutated enzyme or binding polypeptide. Alternatively, the sequence encoding the enzyme or binding polypeptide may be substantially or completely synthetic. For example,
See Goeddel et al., Proc. Natl. Acad. Sci. USA, 76, 106-110, 1979. For the purpose of recombinant expression, codon usage preferences for organisms in which such nucleic acids should be expressed,
It is suitably considered in designing nucleic acids encoding synthetic enzymes or binding polypeptides. For example, a nucleic acid sequence incorporating a prokaryotic codon preference can be
It can be designed from mammalian sequences using a software program such as Oligo-4 available from Bioscience, Inc. (Plymouth, MN).

An embodiment of the nucleic acid sequence of the present invention is preferably a deoxyribonucleic acid sequence, preferably a double-stranded deoxyribonucleic acid sequence. However, they may also be ribonucleic acid sequences.

Numerous methods are known for deleting or mutating a sequence from a nucleic acid sequence encoding a protein and confirming fusion of the protein encoded by such a deleted or mutated sequence. Have been. Accordingly, the present invention also relates to a mutated or deleted version of a nucleic acid sequence encoding a protein that (a) specifically binds to another molecule or (b) retains the intended enzymatic activity. Such analogs may have N-terminal, C-terminal or internal deletions, so long as the appropriate function is retained.

Suitable expression vectors can facilitate the expression of the included polypeptide in a host cell, which can be eukaryotic (including fungi) or proto-antigen. Useful expression vectors include, among others, pRc / CMV (Invitrogen, San Diego, CA),
pRc / RSV (Invitrogen), pcDNA3 (Invitrogen), Zap Exp
less Vector (Stratagene Cloning Systems, LaJolla, CA); pB
k / CMV or pBk-RSV vector (Stratagene), Bluescript II SK +/-
Phagemis Vectors (Stratagene), LacSwitch (Stratagene), pM
AM and pMAM neo (Clontech, Palo Alto, CA), pKSV10 (Phar
macia, Piscataway, NJ), pCRscript (Stratagene) and pCR2.
1 (Invitrogen). Useful yeast expression systems include, for example, pYEU
ra3 (Clontech). Baculovirus vectors useful for expression in insect cells include several viral vectors derived from Invitrogen (San Diego, CA), such as pVL1393, pVL1392, pBluBac2.
, PBluBacHis A, B or C, and pbacPAC6 from Clontech
Is mentioned. Some of such vectors contain an inducible promoter, such as lac
A promoter will be utilized. In one aspect of the invention, an inducible promoter, such as zinc or other metal ion, metabolite or metabolite mimetic,
For example, a promoter that is responsive to isopropylthio-galactoside, or a hormone such as estrogen or ecdysone (eg, found in expression systems available from Invitrogen, San Diego, CA) is desired. Inducible systems help to minimize the deleterious effects that can result from expressed proteins that have toxic effects on expressing cells.

In one aspect of the invention, these polypeptides are preferably mammalian cell lines,
It is preferably expressed in a transformed cell line having an established cell culture history.
In this embodiment, suitable cell lines include COS-1, COS-7, LM (tk ⁻ )
, HeLa, HEK293, CHO, Rat-1 and NIH3T3.

In another embodiment, these polypeptides are realized in a cell line that can be maintained and grown less expensively than a mammalian cell line, such as a bacterial cell line or a fungal cell line, such as a yeast cell line. In this aspect of the invention, E.I. E. coli bacterial cells are very suitable.

In all aspects of the recombination methodology described herein, useful guidance is provided in various prominent books. For example, Sambrook et al., Molecular Cloning: A Laborat
ory Manual, 2nd edition, Cold Spring Harbor Press, 1989; and Ausubel et al., Shor
t Protocols in Molecular Biology, John Wiley & Sons, New York, 1992.

Methods for Producing Recombinant Fusion Proteins One simple method of isolating a polypeptide synthesized under one direction of the nucleic acids of the invention by an organism has a fusion component that is easily affinity purified. Consists in recombinantly expressing a version of the fusion protein. The fusion component may simply be a polypeptide chain that is a member of a binding pair. Components useful for purification include:
For example, glutathione S-transferase encoded on a commercial expression vector (eg, the vector pGEX4T3 available from Pharmacia, Piscataway, NJ). Other useful purification components are, for example, thioredoxin. The glutathione S-transferase-containing fusion protein can be purified on a glutathione affinity column (e.g., available from Pharmacia, Piscataway, New Jersey). If an additional fusion partner is used, the additional fusion partner may be modified by a partial proteolytic digestion approach that preferentially attacks nonstructural regions, such as a linker between the additional fusion partner and the desired fusion protein. Can be removed. These linkers are described, for example, in Chou and Fasman, Biochemistry.
13, 211, 1974 and Chou and Fasman, Adv. In Enzymol. 47, 45-147, 1978. The linker can be designed to include protease target amino acids, such as arginine and lysine residues (amino acids that define the site to be cleaved by trypsin). To construct a linker, a standard synthetic approach to making oligonucleotides may be employed, along with standard subcloning methodologies. GS
Other fusion partners other than T are available. Procedures utilizing eukaryotic cells, especially mammalian cells, are preferred because these cells post-translationally modify proteins and build molecules that are highly similar or identical to native proteins.

Additional purification techniques such as, but not limited to, preparative electrophoresis, FPLC (Pharma
cia, Uppsala, Sweden), HPLC (eg, gel filtration, using reversed phase or slightly hydrophobic columns), gel filtration, differential precipitation (eg, “salting out” precipitation), ion exchange chromatography, and affinity chromatography .

Preferably, the protein is substantially pure, having a purity of at least 20% by weight relative to other proteins, more preferably at least about 50%, even more preferably at least about 70%, even more preferably It is meant to have at least about 90% purity. For the purposes of this application, a fusion protein is said to be "isolated" if it has been separated from other proteins or other macromolecules of the cell or tissue from which it is derived or prepared.

Methods of Preparing Fibrin Compositions In utilizing the fusion proteins of the present invention, blood is drawn, for example, from a patient, and an anticoagulant, eg, trisodium citrate, and a final concentration of about 0.5% w / v. Mix so that Genetically modified mammalian blood and hepatocyte cultures or milk are examples of suitable sources of fibrinogen. Plasma is isolated by centrifugation, which removes the cellular components of blood. The fusion protein is added to the fibrinogen-containing solution, for example, on a molar basis, at a batroxobin concentration of about 0.1 μg / ml to about 100 μg / ml, preferably at a concentration approximately corresponding to a concentration of about 0.5 μg / ml to about 50 μg / ml. Add. A fibrin polymer precipitate is formed from the reaction of fibrinogen with the enzyme incorporated into the fusion protein. When the fibrinogen converting enzyme is batroxobin, the polymer generally consists of fibrin I. The fibrin polymer is isolated by centrifugation or filtration and then in a low pH buffer such as 0.2 M sodium acetate, pH 4.0, preferably at about 2
Dissolve in the presence of 0 mM concentration of calcium ions. Biotin covalently attached to a solid support, such as agarose, is added in an amount sufficient to bind about 99% or more of the fusion protein. The biotinylated support is, for example, Boehringer Manheim (Indian
apolis, IN) or one of the biotinylating agents available from Clontech (Palo Alto, CA) can be prepared by reacting with a solid support having primary amino groups. The biotinylating agent typically has a biotinyl substituent, one to two aminocaproyl spacer groups, and a reactive N-hydroxysuccinimide group.
The solid support can be, for example, an amino-derivatized agarose resin available from Sigma (St. Louis, MO) or an amino-derivatized chromatography matrix available from Pharmacia (Uppsala, Sweden). Solubilized fibrin is removed from the support-bound fusion protein by centrifugation or filtration. This solubilized fibrin is readily available as a hemostatic agent, for example as described in Edwardson et al., European Patent Application 592,242.

Preparation of the Solid Support The solid support to which the second member of the binding pair is attached may be, for example, a carbohydrate-based material such as agarose, cross-linked agarose or cross-linked dextran,
Or made of non-porous material, such as polystyrene beads or non-bead particles. Methods for covalently attaching a molecule to a solid support are well known in the art and include, for example, constructing a reactive site on the solid support with cyanogen bromide, or attaching the solid support to a diglycidyl ether, such as diglycidyl ether. This involves reacting with a bivalent reagent. For example, `` Attachment to Solid Supports '' Means and Feeney, Chemical Mod
ification of Proteins, Holden-Day, San Francisco, 1971, pp. 40-43 or Affi
nity Chromatography: A Practical Approach, Dean et al. IRL Press, Oxford,
See 1991. The disclosures of these two documents are incorporated herein by reference. For coupling with silica-based materials, for example, the alkyloxysilane component can carry the silica reactive component of the divalent coupling agent. For example, reacting gamma-glycidoxypropyltrimethoxysilane with a silica-based material and then reacting it directly with the protein (via the glycidyl ether component)
Alternatively, a second step is employed in which the glycidyl ether is reacted with an amine, followed by the reductive alkylation of the glycoprotein that has been slowly oxidized to contain the aldehyde component (eg, with periodate oxide). A preferred coupling chemistry is to react a carbohydrate-based solid support with a hydrazide group, and then couple the slowly oxidized (eg, with periodate oxide) glycoprotein by reductive alkylation to include an aldehyde component. Ring. Axelsson et al., Thromb. Haemost. 36: 517,
See 1976. The disclosure of which is incorporated herein by reference.

The invention is further described in the following examples, which of course do not limit the invention.

Example 1A-Mammalian vector encoding batroxobin The EcoRI-XbaI fragment encoding batroxobin was ligated with pUC18.
Clone (R & D Systems, Inc., Minneapolis, MN)
It was cloned into the multiple cloning site of I-neo (Promega, UK, Southampton, UK). This expression sequence consists of the sequence of -24 to 228 of batroxobin and includes a leader sequence. Batroxobin enzyme is expressed from a vector obtained in CHO cells.

[0051] Example 1B- Batroxobin - encoding bacterial vector Batroxobin encoding thio reductase fusion protein BsaI-XbaI fragment pCI / ne
o Clone (R & D Systems, Inc., Minneapolis, MN)
It was cloned into the multiple cloning site of TrxFus (Invitrogen). E. FIG.
The resulting fusion protein expressed in E. coli consists of thioredoxin fused at the 5 'end to a 1-228 amino acid sequence of batroxobin via a linker having an enterokinase cleavage site. This fusion protein was purified on a phenylarsine oxide column (Invitrogen, BV, Netherlands).

Example 2 Chemical Formation of a Batroxobin-Avidin Fusion Protein Using N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP, Pierce Chemical Co., Rockford, Ill.) As follows: A covalent complex may be formed between and and avidin: 3 mg of batroxobin with 0.
The reaction was carried out at room temperature for 60 minutes with 1.9 mg of SPDP dissolved in 75 ml of ethanol.
6 mg of avidin was also reacted in a separate container. The protein product of each reaction was run on Sephadex G25 at 50 mM sodium phosphate, 20 mM NaCl,
It was desalted separately with a pH 7.0 buffer. This induced protein is reduced to SP
Activated by exposing the thiol groups derived from DP, mixed together,
The mixed protein was desalted again by gel filtration on Sephadex G25. Batroxobin-avidin conjugate was isolated by gel filtration on Sephadex G100 (from non-conjugated protein).

The sequence listing SEQ ID NO: 1 shows the Gallus gallus cDN for avidin.
A and SEQ ID NO: 2 is its corresponding protein. Amino acids 1-2
4 is believed to be the leader sequence, and amino acids 25-152 are the mature protein. SEQ ID NO: 3 is the cDNA for streptavidin and
ID NO: 4 is the corresponding protein. Amino acids 1 to 24 are believed to be leader sequences, and amino acids 25 to 183 are the mature protein. SEQ ID N
O: 5 is the cDNA of Bostrops atrox of Batroxobin and
ID NO: 6 is the corresponding protein. Amino acids 1-18 are believed to be the leader sequence, and amino acids 25-255 are the mature protein.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07H 21/02 C07K 19/00 4C076 21/04 C12N 1/21 4C084 C07K 19/00 9/48 4H045 C12N 1 / 21 C12P 21/02 C 9/48 C12N 15/00 A C12P 21/02 A61K 37/547 F term (reference) 4B024 AA01 BA14 CA04 DA06 EA04 GA11 GA19 HA01 4B050 CC05 DD11 LL01 4B064 AG01 CA02 CA19 CC24 CE07 CE12 CE14 4B065 AA26X AA91Y AB01 AC14 BA02 BD39 CA33 CA44 4C057 MM02 MM04 MM09 4C076 CC41 CC50 EE41 4C084 AA02 AA03 BA41 BA44 CA53 DC03 NA14 4H045 AA10 AA20 BA10 BA41 CA53 DA89 EA20 EA34 FA74 GA21

Claims (26)

[Claims] 1. A multi-domain protein comprising: a first polypeptide chain comprising fibrinogen converting enzyme; and a second polypeptide chain comprising a first member of a binding pair, wherein: The second polypeptide chain is the first polypeptide chain,
(1) directly through a bond utilizing an N-terminal amino group, C-terminal carboxy group or side chain functional group, (2) via a divalent linking component for linking the group or functional group, or (3) the first poly A multi-domain protein linked by the first member that binds to a second member of the binding pair that is covalently linked to a peptide chain. 2. The multi-domain protein according to claim 1, which is a fusion protein comprising a recombinant protein comprising a contiguous amino acid sequence comprising said second polypeptide chain and said first polypeptide chain. 3. The multi-domain protein according to claim 1, wherein said second polypeptide chain comprises a polypeptide having bithione binding activity. 4. The multidomain protein of claim 3, wherein said second polypeptide chain comprises at least a portion of a streptavidin protein or an avidin protein sufficient to retain biotin binding activity. 5. The streptavidin or avidin protein comprises at least two bition binding sites, and the streptavidin or avidin protein binds to the first polypeptide chain by binding to a biotin molecule. 5. The multi-domain protein of claim 4, wherein said protein is bound to a polypeptide chain. 6. The multi-domain protein of claim 1, wherein said first polypeptide chain comprises a snake venom enzyme. 7. The method according to claim 7, wherein the first polypeptide chain is Aggustrodon actus (Ag
kistrodon acutus), Agkistrodon contortrix contortrix, Agkistrodon haris pallas, Agkistrodon halys pallas, Agkistrodon (caroselasma) rhodostoma (Calloselasma) robostoma rhodost
ps asper), Boslops atrox (B. atrox), Boslops insularis (B. insularis), Boslops jalaraca (B. jararaca), B. Moojeni, Lachesis muta muta , Crotalus adamanteus, Crotalus durissus terrificus, Trimeresurus flavorviridis, Trimeresurus gravirineus
2. The multi-domain protein of claim 1, comprising a snake toxin enzyme from T. gramineus or Bitis gabonica. 8. A recombinant fusion protein comprising a continuous amino acid sequence comprising: a first polypeptide chain comprising fibrinogen converting enzyme; and a second polypeptide chain comprising members of a binding pair. An encoding nucleic acid, wherein said first and second polypeptides are fused directly via a peptide bond or fused via a linker polypeptide chain. 9. A vector comprising the nucleic acid of claim 8 and regulatory sequences sufficient to express the fusion protein in a cell. 10. A cell comprising the vector according to claim 9. 11. A method for producing a fusion protein comprising a fibrinogen converting enzyme and a first member of a binding pair, comprising: growing the cell of claim 10 in a culture medium; and Recovering said fusion protein from the culture medium. 12. A first polypeptide chain comprising fibrinogen converting enzyme; and (a) an amino acid side chain or (b) which can be used to add a plurality of binding partners.
A) an O-linked or N-linked polysaccharide which can be used to add a binding partner;
A recombinant fusion protein comprising a continuous polypeptide chain comprising: a second polypeptide comprising: 13. A method of preparing a fibrin composition, comprising: (1) contacting a composition comprising fibrinogen with an enzyme effective to convert fibrinogen to fibrin, wherein the fibrin composition is formed. Wherein the enzyme comprises 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 comprises the first polypeptide chain. In the polypeptide chain, (1) N-terminal amino group, C
Directly by a bond utilizing a terminal carboxy group or a side chain functional group, or (2) via a divalent linking component linking the group or functional group, or (3) the covalent bond to the first polypeptide chain Being linked by said first member coupled to a second member of the pair; a method comprising: 14. A method for preparing a fibrin composition according to claim 13, further comprising the step of: (2) forming a monomeric fibrin composition from said fibrin composition. 15. The method of preparing a fibrin composition according to claim 14, further comprising: (3) connecting the monomeric fibrin composition to the second of the binding pairs effective to bind the first member. Contacting a solid support to which the members are attached. 16. The method of claim 15, comprising: after the contacting of step (3), (4) removing the solid support and recovering the monomeric fibrin composition thus obtained. 17. An alpha polypeptide chain comprising a snake toxin-derived proteinase effective to convert prothrombin to thrombin, and a beta polypeptide chain which is a member of a binding pair covalently linked to said alpha polypeptide chain. A conjugation protein comprising: 18. The method according to claim 18, wherein the alpha-polypeptide chain is directly linked to the beta-polypeptide chain by (1) bonding using an N-terminal amino group, C-terminal carboxy group or a side chain functional group, or (2) the group or functional group. 18. The conjugated protein of claim 17, wherein the conjugated protein is linked via a functional linking component that links 19. The method according to claim 1, comprising a recombinant protein comprising a contiguous amino acid sequence comprising said alpha polypeptide and said beta polypeptide.
8. The conjugation protein according to 7. 20. A continuous polypeptide comprising: an alpha polypeptide chain comprising a snake toxin-derived proteinase effective to convert prothrombin to thrombin; and a beta polypeptide chain comprising members of a binding pair. A nucleic acid encoding a recombinant fusion protein comprising an amino acid sequence, wherein said alpha and beta polypeptides are fused directly via a peptide bond or fused via a linker polypeptide chain. , Nucleic acids. 21. A vector comprising the nucleic acid of claim 20 and regulatory sequences sufficient to express the fusion protein in a cell. 22. A cell comprising the vector of claim 19. 23. A method for producing a fusion protein comprising a fibrinogen converting enzyme and a first member of a binding pair, wherein the cells of claim 22 are grown in a culture medium; and Recovering said fusion protein from the culture medium. 24. A method of preparing a thrombin composition, comprising: (1) contacting a composition comprising prothrombin with a snake venom enzyme effective to convert prothrombin to thrombin, thus forming a thrombin composition. Wherein the enzyme comprises a multi-domain 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 directly linked to the alpha polypeptide chain by (a) a bond utilizing an N-terminal amino group, a C-terminal carboxy group or a side chain functional group, and (b) a divalent linking the group or functional group. Via a linking moiety or (c) to the second member of the binding pair covalently linked to the alpha polypeptide chain. By the first member for engagement, it is connected, the method. 25. The method of preparing a thrombin composition according to claim 24, further comprising: (2) forming a second member of said binding pair effective for binding said thrombin composition to said first member. Contacting with a solid support to which it is attached. 26. The method according to claim 25, comprising, after the contacting in step (2), (3) removing the solid support and recovering the thrombin composition thus obtained.