Synthesis, purification, and characterization of an Arg sub 152 yields Glu site-directed mutant of recombinant human blood clotting factor VII

Journal Article · Tue Apr 03 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00465a039· OSTI ID:6325391

Wildgoose, P; Kisiel, W ^[1]; Berkner, K L ^[2]

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1. Univ. of New Mexico, Albuquerque (USA)
2. ZymoGenetics, Inc., Seattle, WA (USA)

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 Arg{sub 152}-Ile{sub 153}. 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, the authors 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 Arg{sub 152} {yields} Glu factor VII using a calcium-dependent, murine anti-factor VII monoclonal antibody column. The clotting activity of mutant factor VII was 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. Immunoblots of mutant factor VII with human factor IXa revealed no cleavage, whereas incubation of mutant factor VII with human factor Xa resulted in cleavage of mutant factor VII and the formation of a lower molecular weight degradation product migrating at M{sup r}{approx}40 000. The results are consistent with the proposal that zymogen factor VII possesses no intrinsic proteolytic activity toward factor X or factor IX.

• OSTI ID: 6325391
• Journal Information: Biochemistry; (USA), Vol. 29:13; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
BLOOD COAGULATION FACTORS
MUTAGENESIS
GLYCOPROTEINS
BIOSYNTHESIS
RECOMBINANT DNA
DNA SEQUENCING
ELECTROPHORESIS
MAN
PURIFICATION
ANIMALS
COAGULANTS
DNA
DRUGS
HEMATOLOGIC AGENTS
MAMMALS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PRIMATES
PROTEINS
STRUCTURAL CHEMICAL ANALYSIS
SYNTHESIS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques