Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
2000-06-30
2002-07-02
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C435S212000, C435S226000, C435S214000
Reexamination Certificate
active
06413737
ABSTRACT:
TECHNICAL FIELD
The invention relates to the field of recombinant proteins, more particularly to new variants of the protease ecarin. The invention provides polynucleotides encoding the new ecarin polypeptides, the polypeptides themselves, and methods of using them.
BACKGROUND
A large number of enzymes, particularly proteases, with pathologic effects have been isolated from various snake venoms. Commonly, snake venoms contain procoagulants (e.g., factors which stimulate blood coagulation) and/or hemorrhagic agents (e.g., factors which break down the basement membrane of the vascular endothelium, causing internal bleeding; factors which inhibit platelet aggregation).
The complete amino acid sequences, and in some cases the nucleotide sequences encoding them, have been reported for a number of procoagulants and hemorrhagic agents found in venom from snakes of various genera, including Crotalus, Trimeresus, Lachesis (all of the family Crotalidae), Daboia (Russells' viper), and Echis (Iwanaga et al., 1993, in
Methods in Protein Sequence Analysis
, Imahori et al. eds., pp. 107-115, Plenum Press, London; Tokunaga et al., 1992
, J. Biol. Chem
. 267:14109-14117; Nishida et al., 1995
, Biochem
. 34:1771-1778).
The venom of
Echis carinatus
contains a procoagulant which may be of particular interest for certain segments of the biotechnology industry. The procoagulant, a protease named ecarin which activates prothrombin, is useful in assays of blood coagulation because it is relatively resistant to heparin and other therapeutic anticoagulants (Berry et al., 1998
, Thromb. Haemost
. 79(1):228-233). Ecarin cleaves human prothrombin between residues Arg
320
-Ile
321
to generate meizothrombin. Autocatalytic processing results in the formation of meizothrombin desF1 and then &agr;-thrombin, the mature, active form of thrombin (Rhee et al., 1982
, Biochemistry
21:3437-3443).
Thrombin is a component of a number of hemostat/wound dressing products that are currently in use or in development. Such products generally incorporate active thrombin (e.g., &agr;-thrombin) derived from animal blood, most commonly bovine blood. However, concerns regarding possible disease transmission (especially prion-transmitted diseases) have made animal blood a less attractive source of thrombin. Human blood derived thrombin is also undesirable due to the additional possibility of transmission of viral disease, such as AIDS, hepatitis B, hepatitis C, and the like. Recombinant human thrombin would greatly reduce the risk of disease transmission via thrombin-containing products, as well as the likelihood of adverse immune reactions upon repeat usage of thrombin-containing products. Recombinant thrombin, particularly human recombinant thrombin would, therefore, be a very desirable substitute for blood derived thrombin.
Thrombin is expressed as a “prepro” protein; that is, the protein is expressed including an amino terminal secretion signal sequence (the “pre”domain) as well as a domain which must be removed before the thrombin can be active (the “pro” domain). In vivo prothrombin processing or activation to thrombin is initiated by the formation of the prothrombinase enzyme complex. The prothrombinase complex consists of the enzyme, factor Xa, a cofactor, factor Va, and calcium ions all of which assemble on a phospholipid surface (normally provided by platelets or endothelial cells). Activation can proceed by two possible pathways (the first involves cleavage at Arg271-Thr272, the reaction products being intermediates called fragment 1.2 and prethrombin-2) The fully active prothrombinase complex activates Factor II by cleavage at Arg320-Ile321 yielding the intermediate meizothrombin). A second cleavage gives rise to &agr;-thrombin. The complexity of the natural activation of prothrombin makes it unsuitable for the commercial manufacture of &agr;-thrombin from recombinant prothrombin.
Accordingly, a need exists in the art for compositions and methods that allow the efficient in vitro processing of preprothrombin and prothrombin to active thrombin.
DISCLOSURE OF THE INVENTION
The inventor has found a new form of the enzyme ecarin which has a different sequence from the one known sequence for Kenyan
Echis carinatus
ecarin. The new ecarin was derived from
Echis carinatus leucogaster
, and differs from the known sequence of ecarin by a single amino acid residue. However, the difference between the two sequences is significant, as the alteration is in the catalytic domain of the protein, and involves a change from a proline (in the known sequence) to a serine (in the protein of the invention). Amino acid sequence changes involving proline are considered non-homologous changes, as proline can have drastic effects on secondary and higher protein structure, due to the particular chemical structure of proline.
In one aspect, the invention relates to polypeptides comprising the ecarin metalloprotease domain of SEQ ID NO:1, shown in
FIG. 1
, as well as polynucleotides encoding such proteins. Other polypeptides included within the invention comprise residues 17-616 and/or residues 290-616 of SEQ ID NO:1, shown in
FIG. 1
, as well as polynucleotides encoding the polypeptides. Additionally, the invention provides variants thereof, including variants wherein position 170 of the protein (SEQ ID NO:1) is altered from cysteine to another amino acid residue, such as serine.
In another aspect, the invention relates to methods for making mature thrombin, comprising treating prothrombin with active ecarin of the invention. The thrombin may be treated in vitro with the ecarin of the invention or during production by co-expression of genes encoding the ecarin of the invention and prothrombin in the same cell.
In a further aspect, the invention provides methods of cleaving proteins, such as genetically engineered fusion proteins, containing an ecarin recognition site. The site may be naturally occurring, or the protein may be engineered to contain an ecarin cleavage site.
REFERENCES:
Nishida et al. (1995) Biochemistry 34(5):1771-8.
Olsen David R.
Prior Jeff
Sehl Louis C.
Wallace Donald G.
Cohesion Technologies, Inc.
Osman Richard Aron
Prouty Rebecca E.
Steadman David J.
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