Construction, properties and specific fluorescent labeling of a bovine prothrombin mutant engineered with a free C-terminal cysteine.
Construction, properties and specific fluorescent labeling of a bovine prothrombin mutant engineered with a free C-terminal cysteine.
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用游离 C 末端半胱氨酸改造的牛凝血酶原突变体的构建、特性和特异性荧光标记。
DOI：
10.1093/protein/9.6.545
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Lentz,BR
中科院分区：
文献类型：
--
作者：
Chen,Q;Lord,ST;Lentz,BR
为了确定凝血过程中磷脂酰丝氨酸诱导的构象变化在凝血酶原激活中的作用，构建了重组牛凝血酶原，对其进行表征并显示其具有整体天然样构象。我们引入半胱氨酸来取代先前构建的活性位点突变体的倒数第二个残基（Gly581），并在中国仓鼠卵巢细胞中以0.6μg/ml细胞培养基的水平表达双突变体。荧光素马来酰亚胺的特异性标记是通过二硫苏糖醇的有限还原来实现的，以释放工程化的半胱氨酸，同时保持分子的天然功能特性。标记的平均化学计量为 0.84 探针/蛋白质。探针在 C 末端的位置通过天然凝血酶、大班毒液和羧肽酶 Y 的蛋白水解作用得到证实。双突变体和标记的凝血酶原均可被蛇毒和凝血酶原激活，但正如预期的那样，双突变体半凝血酶不会像天然半凝血酶那样自溶。因此，首次构建了类似天然但经过特殊标记的凝血酶原。该分子将成为阐明凝血酶原激活过程中膜结构作用的重要工具。此外，所描述的方法可能有效地应用于标记其他含二硫键的蛋白质中奇怪的、工程化的半胱氨酸。
To define the role of phosphatidylserine-induced conformational changes in prothrombin activation during blood coagulation, a recombinant bovine prothrombin was constructed, characterized and shown to have a globally native-like conformation. We introduced a cysteine to replace the penultimate residue (Gly581) of a previously constructed active site mutant, and expressed the double mutant in Chinese hamster ovary cells at the level of 0.6 μg/ml of cell culture medium. Specific labeling with fluorescein maleimide was accomplished by limited reduction with dithiothreitol to free the engineered cysteine while maintaining the native-like functional properties of the molecule. The average stoichiometry of labeling was 0.84 probe/protein. The location of the probe at the C-terminus was confirmed by proteolysis by native thrombin, by Taipan venom, and by carboxypeptidase Y. Both the double mutant and labeled prothrombin could be activated by snake venoms and the prothrombinase but, as expected, the double mutant meizothrombin did not autolyze as does native meizothrombin. Thus, for the first time, a native-like but specifically labeled prothrombin has been constructed. This molecule will be an essential tool for elucidating the structural role of membranes during prothrombin activation. In addition, the methods described might be usefully applied to labeling of an odd, engineered cysteine in other disulfide bond-containing proteins.