An efficient refolding method for the preparation of recombinant human prethrombin-2 and characterization of the recombinant-derived alpha-thrombin.
An efficient refolding method for the preparation of recombinant human prethrombin-2 and characterization of the recombinant-derived alpha-thrombin.
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一种有效的重折叠方法，用于制备重组人凝血酶前体 2 和重组衍生的 α-凝血酶的表征。
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发表时间：
2001
期刊：
影响因子：
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通讯作者：
C. Nozaki
中科院分区：
文献类型：
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作者：
K. Soejima;N. Mimura;H. Yonemura;H. Nakatake;T. Imamura;C. Nozaki
在大肠杆菌中制备了重组人凝血酶-2。表达的凝血酶-2前体形成细胞内包涵体，在含有50 mM Tris-HCl、20 mM CaCl(2)、500 mM NaCl、1 mM EDTA、600 mM精氨酸、1 mM半胱氨酸、0.1 mM半胱氨酸、10% （v/v）甘油和0.2% (w/v) Brij-58 （pH为8.5）的缓冲液中，通过简单的一步稀释过程将蛋白重新折叠。再折叠后，用水蛭素基cooh末端肽亲和层析纯化凝血酶前2，再用Echis carinatus蛇毒凝血酶原激活剂（ecarin）活化。然后测试活化蛋白α -凝血酶的几种活性，包括对显色底物的活性、纤维蛋白原纤维蛋白肽A的释放、蛋白C和可由凝血酶激活的纤维蛋白溶解抑制剂的活性、与抗凝血酶的反应性、凝血活性和血小板聚集。动力学数据显示重组α -凝血酶和血浆源性α -凝血酶的活性没有差异。重组人凝血酶-2的产率约为起始溶蛋白量的4-7%。此外，纯化的再折叠蛋白的最终产率为0.5-1%，从1升大肠杆菌细胞培养中可分离出约1 mg的重组凝血酶-2。
Human recombinant prethrombin-2 was produced in Escherichia coli. The expressed prethrombin-2 formed intracellular inclusion bodies from which the protein was refolded by a simple one-step dilution process in buffer consisting of 50 mM Tris-HCl, containing 20 mM CaCl(2), 500 mM NaCl, 1 mM EDTA, 600 mM arginine, 1 mM cysteine, 0.1 mM cystine, 10% (v/v) glycerol, and 0.2% (w/v) Brij-58 at pH 8.5. After refolding, prethrombin-2 was purified by hirudin-based COOH-terminal peptide affinity chromatography, and then activated with Echis carinatus snake venom prothrombin activator (ecarin). The activated protein, alpha-thrombin, was then tested for several activities including activity toward chromogenic substrate, release of fibrinopeptide A from fibrinogen, activation of protein C, and thrombin-activatable fibrinolysis inhibitor, reactivity with antithrombin, clotting activity, and platelet aggregation. The kinetic data showed no differences in activity between our recombinant alpha-thrombin and plasma-derived alpha-thrombin. The yield of refolded recombinant human prethrombin-2 was about 4-7% of the starting amount of solubilized protein. In addition, the final yield of purified refolded protein was 0.5-1%, and about 1 mg of recombinant prethrombin-2 could be isolated from 1 liter of E. coli cell culture.