Study on the extrinsic blood coagulation

在酿酒酵母中表达人组织因子（TF，氨基酸1-217）胞外结构域，利用可诱导酵母酸性磷酸酶启动子和酵母转化酶信号序列引导其分泌到培养液中。纯化了两种活性可溶性形式sTF_< α >（高分子量形式）和stfβ（低分子量形式），产量分别约为10 mg/l和1 mg/l。sds -聚丙烯酰胺凝胶电泳显示sTF_< α >的表观分子质量为150 kDa，每mol蛋白质含有200多个甘露糖残基。sTFbeta的表观分子质量为37 kDa，每mol蛋白质含有22个甘露糖残基。n -糖苷酶F处理使两种rtf的表观分子质量降至35 kDa。sTF_< α >和stfβ的氨基末端序列和氨基酸组成与cDNA序列推断一致，表明分子质量的差异是由低聚糖结构的异质性引起的。在这些重组tf中，stf β对显色底物的viia -酰胺水解活性提高了40倍，对荧光底物的活性提高了147倍，主要影响kcat值。这种增强与从人胎盘中纯化的TF相当。肝素激活的抗凝血酶III抑制了tnf介导的因子viia -酰胺溶解活性增强，形成高分子量复合物。由于用盐酸胍或尿素等变性剂处理stf β导致活性双相丧失，TF的胞外结构域可能由两个离散的结构域组成。该表达系统提供了大量的TF胞外结构域，因此研究其与因子VII的相互作用是可行的。

The extracellular domain of human tissue factor (TF, amino acids 1-217) was expressed in Saccharomyces cerevisiae, using the inducible yeast acid phosphatase promoter and the yeast invertase signal sequence to direct its secretion into the culture broth. Two active soluble forms sTF_<alpha> (high molecular weight form) and sTFbeta (low molecular weight form) were purified, the yield being approximately 10 and 1 mg/liter of culture supernatant, respectively. sTF_<alpha> had an apparent molecular mass of 150 kDa on SDS-polyacrylamide gel electrophoresis and contained more than 200 residues of mannose/mol of protein. sTFbeta had an apparent molecular mass of 37 kDa and contained 22 residues of mannose/mol of protein. N-Glycosidase F treatments of both rTFs reduced the apparent molecular mass to 35 kDa. The amino-terminal sequences and amino acid compositions of sTF_<alpha> and sTFbeta were consistent with those deduced from the cDNA sequence, thereby indicating that the difference in molecular mass is caused by heterogeneity of oligosaccharide structures. Of these recombinant TFs, sTFbeta enhanced factor VIIa-amidolytic activity 40-fold toward the chromogenic substrate and 147-fold toward the fluorogenic substrate, affecting mainly the kcat value. The enhancement was comparable with that of TF purified from human placenta. The TF-mediated enhancement of factor VIIa-amidolytic activity was inhibited by heparin-activated antithrombin III, forming a high molecular weight complex. As treatment of sTFbeta with denaturants such as guanidine hydrochloride or urea led to a biphasic loss of the activity, the extracellular domain of TF probably consists of two discrete domains. This expression system provides a significant amount of the extracellular domain of TF so that studies of interactions with factor VII are feasible.