Expression of completely gamma-carboxylated recombinant human prothrombin.
Human prothrombin cDNA has been expressed in mammalian cells to yield biologically active, fully gamma-carboxylated prothrombin. A 2.0-kilobase cDNA encoding full-length prothrombin was isolated from a human fetal liver library using a cDNA fragment recovered from a lambda gt11 human hepatoma expression library. Prothrombin cDNA was cloned into a mammalian expression vector and transfected into Chinese hamster ovary cells. Selection for expression of dihydrofolate reductase yielded cell lines secreting up to 0.55 microgram/ml of prothrombin. Recombinant prothrombin synthesized in the presence of vitamin K was quantitatively recovered from tissue culture medium by affinity chromatography using conformation-specific antibodies directed against the metal-stabilized, gamma-carboxylated conformer. The purified material migrated as a single band on denaturing polyacrylamide gels with an electrophoretic mobility equivalent to that of plasma-derived human prothrombin. Automated Edman degradation of recombinant prothrombin revealed a single amino-terminal sequence identical to that of plasma-derived prothrombin. Recombinant and plasma-derived prothrombin interacted similarly with antibodies specific for total prothrombin, abnormal des-gamma-carboxyprothrombin, and two metal-stabilized conformers of prothrombin. Recombinant prothrombin exhibited a specific coagulant activity equivalent to that of plasma-derived prothrombin. The gamma-carboxyglutamic acid analysis of recombinant prothrombin demonstrated 9.9 +/- 0.4 mol of gamma-carboxyglutamic acid/mol of prothrombin. These results represent the first description of the expression of a recombinant vitamin K-dependent protein in which all of the expressed protein is gamma-carboxylated.
D015055:1-Carboxyglutamic Acid; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism; D011516:Prothrombin / Q000235:genetics* / Q000378:metabolism; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000378:metabolism*; D013045:Species Specificity
D000906:Antibodies; D000936:Antigen-Antibody Complex; D011994:Recombinant Proteins; D015055:1-Carboxyglutamic Acid; D011516:Prothrombin; D004247:DNA