RECOMBINANT SOLUBLE HUMAN TISSUE FACTOR SECRETED BY SACCHAROMYCES-CEREVISIAE AND REFOLDED FROM ESCHERICHIA-COLI INCLUSION-BODIES - GLYCOSYLATION OF MUTANTS, ACTIVITY AND PHYSICAL CHARACTERIZATION
RECOMBINANT SOLUBLE HUMAN TISSUE FACTOR SECRETED BY SACCHAROMYCES-CEREVISIAE AND REFOLDED FROM ESCHERICHIA-COLI INCLUSION-BODIES - GLYCOSYLATION OF MUTANTS, ACTIVITY AND PHYSICAL CHARACTERIZATION
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DOI：
10.1042/bj3100605
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发表时间：
1995-09-01
影响因子：
4.1
通讯作者：
WRIGHT, PE
中科院分区：
文献类型：
--
作者：
STONE, MJ;RUF, W;WRIGHT, PE
组织因子（TF）是细胞表面跨膜受体，启动外源性和内源性凝血级联反应。TF在三元复合物中与因子VIIa和因子X结合以及使因子VIIa能够蛋白水解活化因子X的能力存在于TF的细胞外结构域中。我们描述了TF（截短TF，tTF）的表面结构域在酿酒酵母和大肠杆菌中的表达和重组蛋白的生化和物理特性。野生型tTF和几种糖基化位点突变体由S.在酵母前原-α-信号序列的控制下，T13 A，N137 D双突变体是以毫克量表达的最均一的变体。野生型tTF在E.大肠杆菌包含体作为具有聚（His）前导序列的融合蛋白。融合蛋白可以完全复性和领导人通过凝血酶的蛋白水解去除，纯化的蛋白质的正确分子量（24729 Da），通过电喷雾质谱确认。重组酵母tTF，E.大肠杆菌和中国仓鼠卵巢细胞在结合因子VIIa、增强因子VIIa的催化活性和增强因子VIIa对因子X的蛋白水解活化的能力方面是相同的。此外，还测定了酵母菌和大肠杆菌的圆二色谱、荧光发射光谱和核磁共振谱.大肠杆菌蛋白表明，这些蛋白质在结构上基本相同。
Tissue factor (TF) is the cell-surface transmembrane receptor that initiates both the extrinsic and intrinsic blood coagulation cascades. The abilities of TF to associate with Factor VIIa and Factor X in a ternary complex and to enable proteolytic activation of Factor X by Factor VIIa reside in the extracellular domain of TF. We describe the expression of the surface domain of TF (truncated TF, tTF) in both Saccharomyces cerevisiae and Escherichia coli and the biochemical and physical characterization of the recombinant proteins. Wild-type tTF and several glycosylation-site mutants were secreted efficiently by S. cerevisiae under the control of the yeast prepro-alpha-signal sequence; the T13A,N137D double mutant was the most homogeneous variant expressed in milligram quantities. Wild-type tTF was expressed in a non-native state in E. coli inclusion bodies as a fusion protein with a poly(His) leader. The fusion protein could be fully renatured and the leader removed by proteolysis with thrombin; the correct molecular mass (24729 Da) of the purified protein was confirmed by electrospray mass spectrometry. Recombinant tTFs from yeast, E. coli and Chinese hamster ovary cells were identical in their abilities to bind Factor VIIa, to enhance the catalytic activity of Factor VIIa and to enhance the proteolytic activation of Factor X by Factor VIIa. Furthermore, CD, fluorescence emission and NMR spectra of the yeast and E. coli proteins indicated that these proteins are essentially identical structurally.