Design of constructs for the expression of biologically active recombinant human factors X and Xa. Kinetic analysis of the expressed proteins.
Activation of vitamin K-dependent plasma proteases occurs by specific interaction with components of the blood coagulation cascade. In this report, we describe the direct expression and enzymatic characterization of the human coagulation zymogen factor X and its activated form, factor Xa, from transformed Chinese hamster ovary fibroblast cell lines. Expression was achieved using either a full-length factor X cDNA or a unique mutant factor Xa cDNA. The functional factor Xa precursor contained a novel tripeptide bridge in place of the native 52-amino acid activation peptide. This mutation allowed for intracellular processing and secretion of the activated form of factor X. Secreted recombinant factors X (rX) and Xa (rXa) were purified by sequential anion-exchange and immunoaffinity chromatography. The enzymatic activities of factors rX and rXa were compared with those of plasma factors X and Xa in three independent assay systems. In comparison to human plasma factor X, the amidolytic, prothrombinase complex, and plasma clotting activities of factor rX were 50, 85, and 43%, respectively. The corresponding comparative activities for factor rXa were 32, 64, and 48%, respectively. The ability to directly express mutant forms of biologically active human factor X will facilitate the structure/function analysis of this important blood coagulation protein and may lead to the development of novel coagulation inhibitors.
Wolf
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COR Therapeutics, Inc., South San Francisco, California 94080.
Sinha
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U
Hancock
T E
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Lin
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PH
Messier
T L
TL
Esmon
C T
CT
Church
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WR
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics; D005170:Factor X / Q000235:genetics* / Q000378:metabolism; D015951:Factor Xa / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011994:Recombinant Proteins
D004792:Enzyme Precursors; D009841:Oligonucleotides; D011994:Recombinant Proteins; D005170:Factor X; D015951:Factor Xa