Jagadeeswaran P, Reddy S V, Haas P
Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio 78284.
Gene. 1990 Feb 14;86(2):279-83. doi: 10.1016/0378-1119(90)90291-x.
The active form of coagulation factor XIII, factor XIIIa, is a transglutaminase that covalently cross-links fibrin molecules by joining gamma-glutamyl and epsilon-lysyl primary amino groups. In this paper we report the design of a yeast expression vector called pPH3 which includes the GAL1-GAL10 promoter, multiple cloning sites, the URA3 selectable marker, the 2 mu sequences and the ampicillin-resistance gene. We have placed full-length factor XIII cDNA into the PstI site of this vector, transformed the yeast cells and expressed human coagulation factor XIII which was found to be enzymatically active based on observations that this recombinant factor XIII: (a) showed immunological identity with placental factor XIII; (b) was demonstrated to have transglutaminase activity, and (c) cross-linked fibrin in a manner identical to human placental factor XIIIa-catalyzed polymerization (gamma-gamma dimerization and alpha polymerization).
凝血因子 XIII 的活性形式，即因子 XIIIa，是一种转谷氨酰胺酶，它通过连接γ-谷氨酰基和ε-赖氨酰伯氨基，使纤维蛋白分子共价交联。在本文中，我们报告了一种名为 pPH3 的酵母表达载体的设计，该载体包括 GAL1 - GAL10 启动子、多克隆位点、URA3 选择标记、2μ 序列和氨苄青霉素抗性基因。我们已将全长因子 XIII cDNA 置于该载体的 PstI 位点，转化酵母细胞并表达人凝血因子 XIII。基于以下观察结果发现该重组因子 XIII 具有酶活性：(a) 与胎盘因子 XIII 具有免疫同一性；(b) 被证明具有转谷氨酰胺酶活性；(c) 以与人类胎盘因子 XIIIa 催化的聚合反应（γ-γ 二聚化和 α 聚合）相同的方式交联纤维蛋白。
