Synthesis, purification, and characterization of an Arg152----Glu site-directed mutant of recombinant human blood clotting factor VII.
Coagulation factor VII circulates in blood as a single-chain zymogen of a serine protease and is converted to its activated two-chain form, factor VIIa, by cleavage of an internal peptide bond located at Arg152-Ile153. Previous studies using serine protease active-site inhibitors suggest that zymogen factor VII may possess sufficient proteolytic activity to initiate the extrinsic pathway of blood coagulation. In order to assess the putative intrinsic proteolytic activity of single-chain factor VII, we have constructed a site-specific mutant of recombinant human factor VII in which arginine-152 has been replaced with a glutamic acid residue. Mutant factor VII was purified in a single step from culture supernatants of baby hamster kidney cells transfected with a plasmid containing the sequence for Arg152----Glu factor VII using a calcium-dependent, murine anti-factor VII monoclonal antibody column. Purified mutant factor VII was indistinguishable from plasma-derived or recombinant wild-type factor VII by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and migrated as a single band with an apparent molecular weight of 50,000. The average specific activity of several mutant factor VII preparations was 0.00025 unit/micrograms, or 0.01% of that observed for recombinant wild-type factor VII preparations. The clotting activity of mutant factor VII was, however, completely inhibited following incubation with dansyl-Glu-Gly-Arg chloromethyl ketone, suggesting that the apparent clotting activity of mutant factor VII was due to a contaminating serine protease.(ABSTRACT TRUNCATED AT 250 WORDS)
Wildgoose
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Department of Pathology, School of Medicine, University of New Mexico, Albuquerque 87131.
Berkner
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Kisiel
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D000590:Amino Acid Chloromethyl Ketones; D001120:Arginine / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003619:Dansyl Compounds; D004591:Electrophoresis, Polyacrylamide Gel; D005167:Factor VII / Q000096:biosynthesis / Q000235:genetics*; D005971:Glutamates / Q000235:genetics*; D018698:Glutamic Acid; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010447:Peptide Hydrolases; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D000590:Amino Acid Chloromethyl Ketones; D003619:Dansyl Compounds; D005971:Glutamates; D011994:Recombinant Proteins; D018698:Glutamic Acid; C030824:dansylglutamyl-glycyl-arginine chloromethyl ketone; D005167:Factor VII; D001120:Arginine; D010447:Peptide Hydrolases