Characterization of Recombinant Human Coagulation Factor XII

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Coagulation factor XII (FXII) is an 80-kDa plasma glycoprotein, which initiates the intrinsic pathway of blood coagulation reactions. FXII is autocleaved after the Arg353 residue upon contact with a negatively c arged surface, to yield active protease FXII (FXIIa). In this study, we prepared PA-tagged recombinant human FXII (PA-hFXII) in a mammalian expression system using Expi293F cells, analyzed the contact-activation response, protease activity, and glycosylation, and compared them to those of human plasma FXII (hFXII). PA-hFXII was purified using affinity chromatography on anti-PA antibodyconjugated Sepharose from the conditioned medium of the transfected Expi293F cells. PA-hFXII was autoactivated when it came in contact with the negatively charged lipid, sulfatide, and its protease activity was found to be similar to that of hFXII. Two potential N-glycosylation sites underwent glycosylation in both, PA-hFXII and hFXII. Oglycosylation in the proline-rich region appeared more homologous in PA-hFXII than in hFXII. These results showed that recombinant PA-hFXII was suitable for the investigation of the molecular mechanism underlying FXII activation.

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