Note: Descriptions are shown in the official language in which they were submitted.
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FIBRINOGEN-CONVERTING ENZYME HYBRIDS
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
5 the binding pair can be used to remove the fission protein from a fibrin
preparation that
was formed through the action of the converting enzyme. The invention is
fixrther
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 fixrther provides a fission protein between a fibrinogen-
converting enzyme
10 and a polypeptide designed to facilitate covalent attachment of one member
of a binding
pair. Also, 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
15 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. In such sealants, 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
20 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.
Generally, 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
25 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.
30 One of the particular advantages of this fibrin sealant of EP 592,242 is
that the
sealant 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
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standard laboratory equipment. Processes for deriving the fibrinogen material
of prior art
sealants are much more demanding and more difl'lcult 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. See, Holm, "Centrifuge Reagent Delivery System", WO
96/16713,
Holm et al., "Method and Device for Separating Fibrin I from Blood Plasma", WO
96/16714 and Holm, "Centrifuge with Annular Filter", WO 96/16715.
The present invention provides additional means to remove the fibrinogen
converting enzyme from the fibrin sealant preparation. In one aspect, the
invention
provides a fusion protein comprising the converting enzyme and another
polypeptide that
can be used to bind the fusion protein to a solid support after the converting
enzyme has
been used to form fibrin from fibrinogen. In another aspect, the invention
provides a
fusion protein comprising the converting enzyme and another polypeptide that
can be
used to covalently attach binding moieties. In yet another aspect, the
converting enzyme
and the other polypeptide are aggregated by means of the other polypeptide
binding to an
member of a binding pair which is covalently linked to the converting enzyme.
Summary of the Invention
In a first embodiment, 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
fimctionalities, (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. In one
embodiment,
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. Such a multivalent
binding entity
can bind to a first polypeptide via a ligand for the binding sites which is
covalently linked
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to the first polypeptide, leaving at least one other binding site available to
bind another
molecule of ligand.
In a second embodiment, 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 fi~sed directly via a peptide bond or fused via
a linker
polypeptide chain.
In a third embodiment, 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. In one
embodiment,
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
In a fourth embodiment, 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 fi~nctionalities, (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. Preferably, where the
fibrin
composition is not a monomeric fibrin composition, the method further
comprises: (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 having bound thereto a second member of the binding pair effective to
bind the
first member. In another preferred aspect, the method fizrther comprises after
the
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contacting of step (3): (4) removing the solid support and recovering a
resulting
monomeric fibrin composition.
In a fifth embodiment, 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.
In a sixth embodiment, the invention provides a nucleic acid encoding a
recombinant fission 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
fi~sed via a linker polypeptide chain.
In a seventh embodiment, 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.
Preferably, the method fi~rther 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. In another preferred aspect, the method further comprises:
(3)
removing the solid support and thereby recovering a resulting thrombin
composition.
Definitions
~ alpha, beta, first, second: Terms such as "first", "second", "alpha" and
"beta" are
used herein as arbitrary names to help distinguish recitals of similar
elements.
~ bifunctional linkage group: A bifunctional linkage group is a molecule
having two
sites for attaching to a protein or polypeptide. Preferred bifixnctional
linkage groups are
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polypeptides of from one to about 3 amino acids, preferably of from one to
about 30
amino acids. Additionally, 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.
~ fibrinogen-converting enzyme: A fibrinogen-converting enzyme is a substance
that
catalyzes a conversion of fibrinogen to a derivative that spontaneously
polymerizes
noncovalently to form fibrin polymer. Generally, the derivative will be fibrin
I (desAA-
fibrin), fibrin II (desAA-desBB-fibrin) or desBB-fibrin.
~ high affinity binding: 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 ai~nity ligand for the isolation of the other substance. Typically,
high affinity
binding is reflected in a association constant of about 105 M-1 or more,
preferably 106
M-1 or more; more 10~ M-1 or more.
~ monomeric fibrin: 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 ultracentrifixgation or gel filtration substantially all of
the fibrin chain
molecules in the composition behave as the non-polymerized hexa.mers
(a2~i2Y2)~ The
phrase "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%.
For solid
forms, 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. If necessary, more involved analytical techniques
can be
applied to ascertain if the fibrin hexamers are not aggregated with polymer-
forming non-
covalent bonds of type and strength involved in the formation of fibrin clots.
Solid forms
can include suspended solids within a liquid in which the fibrin is not
soluble, such as a
suspension in acetone.
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~ 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.
~ recombinant protein: 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.
~ snake venom-derived proteinase: 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.
Detailed Description
Enz~rme-to Binding Polypeptide Fusion Protein
The fibrinogen-converting enzyme is preferably batroxobin ("Btx"), i.e., a
proteinase from the snake venom of snakes of the genus Bothrops. 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. In one embodiment, enzymes from Bothrops are used.
The sequence for a fibrinogen-converting enzyme from Agkistrodon rhodostoma
is described in B ach et al., W090/063 62. Two sequences from Agkristrodon c.
contortrix
are described in Valenzuela et al., EP O 323 722. The sequence of batroxobin
(from
Bothrops atrox moojeni), is described in Japanese Patent Application (Kokai) 2-
124092.
Sequences for batroxobin, an enzyme from Trimeresurus jlavorviridis, and an
enzyme
from Crotalus horridus are reported in Pirkle and Theodor, "Structure of
Thrombin-like
snake venom Proteinases", in Medical use of Snake Venom Proteins. Sequences
from
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Agkristrodon c. contortix, Russell's viper, Bothrops atrox moojeni and
Trimeresurus
jZavoviridis are described in McMullen et al., Biochemistry 28:674-679, 1989.
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
(Vipers Russelli) are available from the SWISS-PROT protein database
(accessible
through the NCBI) under accession numbers P18964 and P18965. 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 I06680, I06681, I06724 and 106751.
When the fibrinogen-converting enzyme is thrombin, generally the fusion
protein
of the invention will be formed by chenvcal 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
W091/11519
("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
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prothrombin-converting enzyme is preferably from the venom of Ecchis
carinatus. Other
snake venom proteinases that are suitable, include without limitation the
venom enzymes
from Australian tiger snake and Akistrodon hadlys pallys. For example, the
sequence for
a prothrombin activator (ecarin) from Kenyan Echis carinatus venom is
described in
5 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 Seguence, Vol.
5, National
Biomedical Research Foundation, Washington, DC, 1972 (see also, DeLange and
Huang,
10 J. Biol. Chem. 246: 698-709, 1971 ), and an amino acid sequence for
Streptavidin is
described in Argarana et al., Nucl. Acid Res. 14:1871-1882, 1986. Nucleic acid
sequences are available, for example, as follows: (I) chicken mRNA for avidin,
Gene
Bank Acc. No. X5343, Gore et al., Nucl. AcidRes. 15: 3595-3606, 1987; (2)
chicken,
strain White Leghorn mRNA for avidin, Gene Bank Acc. No. L27818 (3)
streptavidin
15 from Strep. avidinii, Gene Bank Acc. No. X03591, Argarana et al., Nucl.
Acid Res.
14:1871-1882, 1986; (4) synthetic gene for streptavidin from Strep. avidinii,
Gene Bank
Acc. No. A00743, Edwards, W089103422; 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 tetrameric form, although
20 monomers can be used. In reciting herein that 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. Other useful members of a 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
25 (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
30 Biotech, Piscataway, NJ). Methods of producing such antibodies are
available to those of
ordinary skill in Iight of the ample description herein of polypeptide
expression systems
and of known antibody production methods. For antibody preparation methods,
see, for
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example, Ausubel et al., Short Protocols in Molecular Biology, John Wiley &
Sons, New
York, 1992. The binding-pairs used in the invention preferably display high
affinity
binding even at relatively low pH, such as a pH of about 5. In some
embodiments of the
invention, the member of a binding pair that is attached to an enzyme is not
limited to
S polypeptide members of binding pairs. In this case, biotin is the most
preferred such
member of a binding pair.
In many embodiments of the invention, 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:
1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr,
Pro and Gly;
2. Polar, negatively charged residues and their amides: Asp, Asn, Glu
and Gln;
3. Polar, positively charged residues: His, Arg and Lys;
4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and
5. Aromatic residues: Phe, Tyr and Trp.
A preferred listing of conservative variations is the following:
Original Residue Variation
Ala Gly, S er
Arg Lys
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Asn Gln, His
Asp Glu
Cys Ser
Gln Asn
Glu Asp
Gly Ala, Pro
His Asn, Gln
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln,
Glu
Met Leu, Tyr,
Ile
Phe Met, Leu,
Tyr
Ser Thr
Thr S er
Trp Tyr
Tyr Trp, Phe
Val Ile, Leu
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-
s 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.
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 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
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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.
In other embodiments, 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 bifimctional crosslinking reagents. Such
bifunctional reagents include compounds with activated acyl esters such as N-
hydroxysuccinimide esters, mercuric ion, other mercury compounds, compounds
containing maleimide fianctionalities, 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.
Among the modifications that can be added to a protein by recombinant methods
are sequences that are glycosylated when the protein is expressed in an
appropriate cell.
N-linked glycosylations typically occur at the asparagine of Asn-Xaa-Ser/Thr
tripeptide
subsequences of glycoproteins.
Conversing Enzyme Attachable Polypeptide Fusion Protein
In one embodiment of the invention, 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. Such 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 usefix! in linking the member of a binding pair.
Preferably, the
facilitative polypeptide comprises between about 10 and about 50 amino acid
residues,
more preferably between about 20 and about 30 amino acid residues. Preferably,
the
linkable amino acid comprises lysine, arginine, histidine, aspartic acid,
glutamic acid or
cysteine, more preferably lysine or cysteine, and yet more preferably lysine.
For methods
on how to link biotin to the facilatitive polypeptide can be found, for
example, in Savage
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et al., Avidin. Biotin Chemistry: A Handbook, Pierce Chemical Co., 1992. 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. Cells and Methods
Where nucleic acid sequence is known, or sufficient amino acid sequence is
known
to predict useful primers, 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. 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. One such method is referred to as PCR-
RACE,
and protocols for this method are available, for example, from Gibco BRL
(Gaithersburg,
MD). Where 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. Where restriction sites are designed into the PCR
primers, it
can be necessary to change the colons used to encode particular amino acid
residues or to
make mutational changes (preferably conservative) to design the restriction
site. In some
20 cases, it may be necessary to subclone a particular nucleic acid fragment
and use well-
established site directed mutagenesis techniques to engineer the needed
restriction sites.
See, Ausubel et al, Current Protocols in Molecular Biology, John Wiley and
Sons, New
York, 1995, pp.8.1.1-8.1.6.
To construct non-naturally occurnng enzyme- or binding polypeptide-encoding
nucleic acids, the native sequences can be used as a starting point and
modified to suit
particular needs. For instance, 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
30 ligation reactions. The cassettes can be used to substitute synthetic
sequences encoding
mutated enzyme or binding polypeptide amino acid sequences. Alternatively, the
enzyme
or binding polypeptide-encoding sequence can be substantially or fully
synthetic. See, for
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-13-
example, Goeddel et al., Proc. Natl. Acad. Sci. USA, 76, 106-110, 1979. For
recombinant expression purposes, 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. For example, 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.
Numerous methods are known to delete sequence from or mutate nucleic acid
sequences that encode a protein.and to confirm the function of the proteins
encoded by
these deleted or mutated sequences. Accordingly, 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
fi~nction is retained.
A suitable expression vector is capable of fostering expression of the
included
polypeptide in a host cell, which can be eukaryotic (including fiangal), or
prokaryotic.
Useful expression vectors include pRc/CMV (Invitrogen, San Diego, CA), pRcIRSV
(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), pKSV 10 (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 pbacPAC6 from Clontech. Some of
these vectors will utilize inducible promoters such as the lac promoter. In
one aspect of
the invention, 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
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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.
In one embodiment of the invention, the polypeptides are preferably expressed
in a
mammalian cell line, preferably a transformed cell line with 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, 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. In this aspect of the
invention, E. coli
bacterial cells are particularly preferred.
In all aspects of recombinant methodology referred to herein, ample guidance
can
be found in a number of widely recognized authorities including: Sambrook et
al.,
Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press,
1989;
15 and Ausubel et al., Short Protocols in Molecular Biology, John Wiley &
Sons, New
York, 1992.
Methods o~'Producing Recombinant Fusion Proteins
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 fi~sed 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 fizsion protein can then be purified on a glutathione
affinity
column (for instance, that available from Pharmacia, Piscataway, New Jersey).
If extra
fission partners are used, the extra fusion partner can be removed by partial
proteolytic
digestion approaches that preferentially attack unstructured regions such as
the linkers
30 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
CA 02312476 2000-06-OS
WO 99129838 PCTIUS98126086
-15-
potential developed, for instance, by Chou and Fasman, Biochemistry 13, 21 l,
1974 and
Chou and Fasman, Adv. in Enrymol. 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. To create the linkers,
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.
Additional purification techniques can be applied, including without
limitation,
preparative electrophoresis, FPLC (Pharmacia, Uppsala, Sweden), HPLC (e.g.,
using gel
filtration, reverse-phase or mildly hydrophobic columns), gel filtration,
differential
precipitation (for instance, "salting out" precipitations), ion-exchange
chromatography
and afF~nity chromatography.
Preferably, 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%. For the purposes of
this
application, 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.
Methods of Preparing Fibrin Compositions
In using the fusion protein of the invention, 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. Blood and liver cell cultures, recombinant cultures or milk
from
transgenically modified mammals are examples of suitable sources of
fibrinogen. 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 pglml to about 100 pg/ml, preferably to a
concentration of
about 0.5 pg/ml to about 50 pg/ml. A precipitate of fibrin polymer forms from
the
reaction of fibrinogen with enzyme incorporated into the fusion protein. Where
that
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
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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. Biotin covalently bound
to a solid
support such as agarose is added in sufficient quantity to bind in excess of
about 99% of
the fusion protein. The biotinylated support can be prepared, for example, by
reacting
S 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 oaf 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 are
well known
in the art, and 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 Modifrcation of Proteins, Holden-Day, San Francisco, 1971,
pp. 40-43
or 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. For coupling with silica-based materials,
alkyloxysilane moieties,
for example, can provide the silica-reactive moiety of a bifunctional coupling
reagent. For
example, y-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
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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.
The following examples further illustrate the present invention, but of
course,
S should not be construed as in any way limiting its scope.
Example 1 A - Mammalian Vector Encoding Batroxobin
A EcoRT-XbaI fragment encoding batroxobin is excised from a pUC 18 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 1B - Bacterial Vector Encoding Batroxobin-thioreductase fusion Protein
A BsaI-XbaI 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 S' 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 NaCI, 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 6100.
Sequence Listing
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SEQ ID NO:1 is of the Gallus gallars cDNA for avidin, and SEQ ID N0:2 is the
corresponding protein. Amino acids 1-24 are believed to be the leader
sequence, and
amino acids 25-152 the mature protein. SEQ LD N0:3 is an cDNA for
streptavidin, and
SEQ ID N0:4 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:S is of
the
Bothrops atrox cDNA for batroxobin, and SEQ ID N0:6 is the corresponding
protein.
Amino acids 1-18 are believed to be the leader sequence, and amino acids 25-
255 the
mature protein.
All publications and references, including but not limited to patents and
patent
applications, cited in this specification are herein incorporated by reference
in their
entirety as if each individual publication or reference were specifically and
individually
indicated to be incorporated by reference herein as being fully set forth. Any
patent
application to which this application claims priority is also incorporated by
reference
herein in its entirety in the manner described above for publications and
references.
1 S While this invention has been described with an emphasis upon preferred
embodiments, it will be obvious to those of ordinary skill in the art that
variations in the
preferred devices and methods may be used and that it is intended that the
invention may
be practiced otherwise than as specifically described herein. Accordingly,
this invention
includes all modifications encompassed within the spirit and scope of the
invention as
defined by the claims that follow.
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A- 1
SEQUENCE LISTING
<110> Bristol-Myers Squibb Company
Stewart A. Cederhom-Williams
<120> Fibrinogen-Converting Enzyme Hybrids
<130> CV0268
<150> 60/067,978
<151> 1997-12-09
<160> 6
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 459
<212> DNA
<213> Gallus gallus
<220>
<221> CDS
<222> (1)...(456)
<900> 1
atggtgcac gcaacc tccccgctg ctgctg ctgctg ctgctcagc ctg 48
MetValHis AlaThr SerProLeu LeuLeu LeuLeu LeuLeuSer Leu
1 5 10 15
getctggtg getccc ggcctctct gccaga aagtgc tcgctgact ggg 96
AlaLeuVal AlaPro GlyLeuSer AlaArg LysCys SerLeuThr Gly
20 25 30
aaatggacc aacgat ctgggctcc aacatg accatc ggggetgtg aac 149
LysTrpThr AsnAsp LeuGlySer AsnMet ThrIle GlyAlaVal Asn
35 40 45
agcagaggt gaattc acaggcacc tacatc acagcc gtaacagcc aca 192
SerArgGly GluPhe ThrGlyThr TyrIle ThrAla ValThrAla Thr
50 55 60
tcaaatgag atcaaa gagtcacca ctgcat gggaca caaaacacc atc 240
SerAsnGlu IleLys GluSerPro LeuHis GlyThr GlnAsnThr Ile
65 70 75 80
aacaagagg acccag cccaccttt ggcttc accgtc aattggaag ttt 288
AsnLysArg ThrGln ProThrPhe GlyPhe ThrVal AsnTrpLys Phe
85 90 95
tcagagtcc accact gtcttcacg ggccag tgcttc atagacagg aat 336
SerGluSer ThrThr ValPheThr GlyGln CysPhe IleAspArg Asn
100 105 110
gggaaggag gtcctg aagaccatg tggctg ctgcgg tcaagtgtt aat 384
GlyLysGlu ValLeu LysThrMet TrpLeu LeuArg SerSerVal Asn
115 120 125
gacattggt gatgac tggaaaget accagg gtcggc atcaacatc ttc 432
AspIleGly AspRsp TrpLysAla ThrArg ValGly IleAsnIle Phe
130 135 140
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A-2
act cgc ctg cgc aca cag aag gag tga 459
Thr Arg Leu Arg Thr Gln Lys Glu
145 150
<210> 2
<211> 152
<212> PRT
<213> Gallus gallus
<400> 2
Met Val His Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu Ser Leu
1 5 10 15
Ala Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly
20 25 30
Lys Trp Thr Asn Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn
35 90 45
Ser Arg Gly Glu Phe Thr Gly Thr Tyr Ile Thr Ala Val Thr Ala Thr
50 55 60
Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr Gln Asn Thr Ile
65 70 75 80
Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys Phe
85 90 95
Ser Glu Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile Asp Arg Asn
100 105 110
Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu Arg Ser Ser Val Asn
115 120 125
Asp Ile Gly Asp Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile Phe
130 135 140
Thr Arg Leu Arg Thr Gln Lys Glu
145 150
<210> 3
<211> 552
<212> DNA
<213> Streptococcus
<220>
<221> CDS
<222> (1)...(549)
<900> 3
atg cgc aag atc gtc gtt gca gcc atc gcc gtt tcc ctg acc acg gtc 48
Met Arg Lys Ile Val Val Ala Ala Ile Ala Val Ser Leu Thr Thr Val
1 5 10 15
tcg att acg gcc agc get tcg gca gac ccc tcc aag gac tcg aag gcc 96
Ser Ile Thr Ala Ser Ala Ser Ala Asp Pro Ser Lys Asp Ser Lys Ala
20 25 30
cag gtc tcg gcc gcc gag gcc ggc atc acc ggc acc tgg tac aac cag 144
Gln Val Ser Ala Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln
35 40 45
ctc ggc tcg acc ttc atc gtg acc gcg ggc gcc gac ggc gcc ctg acc 192
Leu Gly Ser Thr Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr
50 55 60
gga acc tac gag tcg gcc gtc ggc aac gcc gag agc cgc tac gtc ctg 240
Gly Thr Tyr Glu Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu
65 70 75 80
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A-3
accggtcgttac gacagc gccccggcc accgac ggcagcggc accgcc 288
ThrGlyArgTyr AspSer AlaProAla ThrAsp GlySerGly ThrAla
85 90 95
ctcggttggacg gtggcc tggaagaat aactac cgcaacgcc cactcc 336
LeuGlyTrp-ThrValAla TrpLysAsn AsnTyr ArgAsnAla HisSer
100 105 110
gcgaccacgtgg agcggc cagtacgtc ggcggc gccgaggcg aggatc 384
AlaThrThrTrp SerGly GlnTyrVal GlyGly AlaGluAla ArgIle
115 120 125
aacacccagtgg ctgctg acctccggc accacc gaggccaac gcctgg 432
AsnThrGlnTrp LeuLeu ThrSerGly ThrThr GluAlaAsn AlaTrp
130 135 140
aagtccacgctg gtcggc cacgacacc ttcacc aaggtgaag ccgtcc 480
LysSerThrLeu ValGly HisAspThr PheThr LysValLys ProSer
145 150 155 160
gccgcctccatc gacgcg gcgaagaag gccggc gtcaacaac ggcaac 528
AlaAlaSerIle AspAla AlaLysLys AlaGly ValAsnAsn GlyAsn
165 170 I75
ccgctcgacgcc gttcag cagtag 552
ProLeuAspAla ValGln Gln
180
<210> 4
<211> 183
<212> PRT
<213> Streptococcus
<400> 4
MetArg Ile ValVal AlaAlaIle AlaVal LeuThr ThrVal
Lys Ser
1 5 10 15
SerIle Ala SerAla SerAlaAsp ProSer AspSer LysAla
Thr Lys
20 25 30
GlnVal Ala AlaGlu AlaGlyIle ThrGly TrpTyr AsnGln
Ser Thr
35 90 45
LeuGly Thr PheIle ValThrAla GlyAla GlyAla LeuThr
Ser Asp
50 55 60
GlyThr Glu SerAla ValGlyAsn AlaGlu ArgTyr ValLeu
Tyr Ser
65 70 75 80
ThrGly Tyr AspSer AlaProAla ThrAsp SerGly ThrAla
Arg Gly
85 90 95
LeuGly Thr ValRla TrpLysAsn AsnTyr AsnAla HisSer
Trp Arg
100 105 110
AlaThr Trp SerGly GlnTyrVal GlyGly GluAla ArgIle
Thr Ala
115 120 125
AsnThr Trp LeuLeu ThrSerGly ThrThr AlaAsn AlaTrp
Gln Glu
130 135 140
LysSer Leu ValGly HisAspThr PheThr ValLys ProSer
Thr Lys
195 150 155 160
AlaAla Ile AspAla AlaLysLys AlaGly AsnAsn GlyAsn
Ser Val
165 170 175
ProLeu Ala ValGln Gln
Asp
180
<210> 5
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A- 4
<2 11>768
<2 12>DNA
<2 13>Bothropsatrox
<2 20>
<2 21>CDS
<2 22>(1)...(765)
<4 00>5
atggtg ctgatc agagtgata gcaaac cttctgata ttacag gtttct 48
MetVal LeuIle ArgValIle AlaAsn LeuLeuIle LeuGln ValSer
1 5 10 15
tacgca caaaag tcttctgaa ctggtc attggaggt gatgaa tgtgac 96
TyrAla GlnLys SerSerGlu LeuVal IleGlyGly AspGlu CysAsp
20 25 30
ataaat gaacat cctttcctt gcattc atgtactac tctccc cggtat 144
IleAsn GluHis ProPheLeu AlaPhe MetTyrTyr SerPro ArgTyr
35 40 45
ttctgt ggtatg actttgatc aaccag gaatgggtg ctgacc getgca 192
PheCys GlyMet ThrLeuIle AsnGln GluTrpVal LeuThr AlaAla
50 55 60
cactgt aacagg agatttatg cgcata caccttggt aaacat gccgga 240
HisCys AsnArg ArgPheMet ArgIle HisLeuGly LysHis AlaGly
65 70 75 80
agtgta gcaaat tatgatgag gtggta agataccca aaggag aagttc 288
SerVal AlaAsn TyrAspGlu ValVal ArgTyrPro LysGlu LysPhe
85 90 95
atttgt cccaat aagaaaaaa aatgtc ataacggac aaggac attatg 336
IleCys ProAsn LysLysLys AsnVal IleThrAsp LysAsp IleMet
100 105 110
ttgatc aggctg gacagacct gtcaaa aacagtgaa cacatc gcgcct 384
LeuIle ArgLeu AspArgPro ValLys AsnSerGlu HisIle AlaPro
115 120 125
ctcagc ttgcct tccaaccct cccagt gtgggctca gtttgc cgtatt 432
LeuSer LeuPro SerAsnPro ProSer ValGlySer ValCys ArgIle
130 135 140
atggga tggggc gcaatcaca acttct gaagacact tatccc gatgtc 480
MetGly TrpGly AlaIleThr ThrSer GluAspThr TyrPro AspVal
145 150 155 160
cctcat tgtget aacattaac ctgttc aataatacg gtgtgt cgtgaa 528
ProHis CysAla AsnIleAsn LeuPhe AsnAsnThr ValCys ArgGlu
165 170 175
gettac aatggg ttgccggcg aaaaca ttgtgtgca ggtgtc ctgcaa 576
AlaTyr AsnGly LeuProAla LysThr LeuCysAla GlyVal LeuGln
180 185 190
ggaggc atagat acatgtggg ggtgac tctggggga cccctc atctgt 624
GlyGly IleAsp ThrCysGly GlyAsp SerGlyGly ProLeu IleCys
195 200 205
aatgga caattc cagggcatt ttatct tggggaagt gatccc tgtgcc 672
CA 02312476 2000-06-OS
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A-5
Asn Gly Gln Phe Gln Gly Ile Leu Ser Trp Gly Ser Asp Pro Cys Ala
210 215 220
gaa ccg cgt aag cct gcc ttc tac acc aag gtc ttt gat tat ctt ccc 720
Glu Pro Arg Lys Pro Ala Phe Tyr Thr Lys Val Phe Asp Tyr Leu Pro
225 230 235 240
tgg atc cag agc att att gca gga aat aaa act gcg act tgc ccg 765
Trp Ile Gln Ser Ile Ile Ala Gly Asn Lys Thr Ala Thr Cys Pro
245 250 255
tga 768
<210> 6
<211> 255
<212> PRT
<213> Bothrops atrox
<400> 6
Met Val Leu Ile Arg Val Ile Ala Asn 'Leu Leu Ile Leu Gln Val Ser
1 5 10 15
Tyr Ala Gln Lys Ser Ser Glu Leu Val Ile Gly Gly Asp Glu Cys Asp
20 25 30
Ile Asn Glu His Pro Phe Leu Ala Phe Met Tyr Tyr Ser Pro Arg Tyr
35 90 45
Phe Cys Gly Met Thr Leu Ile Asn Gln Glu Trp Val Leu Thr Ala Ala
50 55 60
His Cys Asn Arg Arg Phe Met Arg Ile His Leu Gly Lys His Ala Gly
65 70 75 80
Ser Val Ala Asn Tyr Asp Glu Val Val Arg Tyr Pro Lys Glu Lys Phe
85 90 95
Ile Cys Pro Asn Lys Lys Lys Asn Val Ile Thr Asp Lys Asp Ile Met
100 105 110
Leu Ile Arg Leu Asp Arg Pro Val Lys Asn Ser Glu His Ile Ala Pro
115 120 125
Leu Ser Leu Pro Ser Asn Pro Pro Ser Val Gly Ser Val Cys Arg Ile
130 135 140
Met Gly Trp Gly Ala Ile Thr Thr Ser Glu Asp Thr Tyr Pro Asp Val
145 150 155 160
Pro His Cys Ala Asn Ile Asn Leu Phe Asn Asn Thr Val Cys Arg Glu
165 170 175
Ala Tyr Asn Gly Leu Pro Ala Lys Thr Leu Cys Ala Gly Val Leu Gln
180 185 190
Gly Gly Ile Asp Thr Cys Gly Gly Asp Ser Gly Gly Pro Leu Ile Cys
195 200 205
Asn Gly Gln Phe Gln Gly Ile Leu Ser Trp Gly Ser Asp Pro Cys Ala
210 215 220
Glu Pro Arg Lys Pro Ala Phe Tyr Thr Lys Val Phe Asp Tyr Leu Pro
225 230 235 240
Trp Ile Gln 5er Ile Ile Ala Gly Asn Lys Thr Ala Thr Cys Pro
245 250 255
