Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin.
Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin.
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重组α-凝血酶的制备：重组人凝血酶前体2的高水平表达及其重组ecarin的激活。
DOI：
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
C. Nozaki
中科院分区：
文献类型：
--
作者：
H. Yonemura;T. Imamura;K. Soejima;Y. Nakahara;W. Morikawa;Y. Ushio;Yasuharu Kamachi;H. Nakatake;K. Sugawara;T. Nakagaki;C. Nozaki
我们已经建立了大规模生产重组人α-凝血酶的生产系统。在该系统中，高产率的α-凝血酶是由重组ecarin激活的凝血酶原-2制备的。我们利用小鼠骨髓瘤细胞和携带鸡β-肌动蛋白启动子和突变型二氢叶酸还原酶基因的表达载体，生产了人凝血酶前2。为了进一步提高凝血酶原-2的表达，我们在50升悬浮培养中加入植物蛋白胨进行补料分批培养。经5次植物蛋白胨培养后，重组凝血酶原-2的表达水平达到200微克/毫升。随后，以类似方式表达的重组凝血酶原-2可被激活为α-凝血酶。最后，重组α-凝血酶经苯甲酰胺-琼脂糖凝胶亲和层析纯化为均一组分。在培养基中凝血酶原-2的产率约为70%。纯化的重组α-凝血酶的活性，包括对发色底物的水解、纤维蛋白肽A的释放和蛋白C的激活，与血浆来源的α-凝血酶没有区别。本系统适用于大规模生产重组α-凝血酶，可替代临床上可用的人或牛血浆来源的α-凝血酶。
We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.
影响因子：
2.9
作者：
DAVIE, EW;FUJIKAWA, K;KISIEL, W
通讯作者：
KISIEL, W
DOI：
10.1016/s0021-9258(18)34900-7
发表时间：
1982-03
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
D. Tollefsen;D. Majerus;M. Blank
通讯作者：
D. Tollefsen;D. Majerus;M. Blank
人工心脏瓣膜患者手术后抗凝血酶抗体的产生。
影响因子：
20.3
作者：
Stricker,RB;Lane,PK;Leffert,JD;Rodgers,GM;Shuman,MA;Corash,L
通讯作者：
Corash,L