Board P G, Pierce K, Coggan M
Human Genetics Group, John Curtin School of Medical Research, Australian National University, Canberra.
Thromb Haemost. 1990 Apr 12;63(2):235-40.
Coagulation factor XIII is a zymogen that can be activated by thrombin cleavage to a transglutaminase that catalyses the formation of covalent crosslinks between fibrin chains in the final stages of the blood clotting cascade. Although circulating factor-XIII is composed of A and B subunits the catalytic activity is a property of the A subunits. In this study we have constructed a plasmid (pKKF13A) that contains a cDNA encoding the A subunit positioned downstream of a tac promoter. Escherichia coli containing this plasmid produce A subunit protein when grown in the presence of IPTG. The cloned A subunit has been partially purified and characterized. Comparison with A subunits purified from plasma showed that the cloned A subunits were of the same size, assembled as dimers, and had the same native electrophoretic mobility. The cloned A subunits expressed transglutaminase activity with putrescine, dansylcadaverine and casein as substrates, and were able to crosslink fibrin in clots formed from A subunit deficient plasma. These studies have demonstrated that functional recombinant factor XIII A subunit can be produced in E. coli and suggest that recombinant factor XIII can potentially provide a safe and inexhaustible supply for therapeutic use.
凝血因子 XIII 是一种酶原，可被凝血酶切割激活成为转谷氨酰胺酶，该酶在血液凝固级联反应的最后阶段催化纤维蛋白链之间共价交联的形成。虽然循环中的因子 XIII 由 A 和 B 亚基组成，但催化活性是 A 亚基的特性。在本研究中，我们构建了一个质粒（pKKF13A），其包含一个编码 A 亚基的 cDNA，该 cDNA 位于 tac 启动子的下游。含有此质粒的大肠杆菌在 IPTG 存在下生长时会产生 A 亚基蛋白。已对克隆的 A 亚基进行了部分纯化和表征。与从血浆中纯化的 A 亚基比较表明，克隆的 A 亚基大小相同，组装成二聚体，并且具有相同的天然电泳迁移率。克隆的 A 亚基以腐胺、丹磺酰尸胺和酪蛋白为底物表现出转谷氨酰胺酶活性，并且能够在由缺乏 A 亚基的血浆形成的凝块中交联纤维蛋白。这些研究表明，功能性重组因子 XIII A 亚基可以在大肠杆菌中产生，并表明重组因子 XIII 有可能为治疗用途提供安全且取之不尽的供应。
