Department of Biological Science, Myongji University, Yongin 449-728, Korea.
J Microbiol Biotechnol. 2010 Jan;20(1):132-7.
The skc gene encoding streptokinase (SK), with a molecular weight of approximately 47.4 kDa, was cloned from Streptococcus eouisimilis ATCC9542 and heterologously overexpressed in Streptomyces lividans TK24 and E. coli using various strong promoters. When the sprT promoter was used in the S. lividans TK24 host, the SK protein corresponding to 47.4 kDa was detected with a smaller hydrolyzed protein (44 kDa), implying posttranslational hydrolysis occurred as reported in other expression systems. Casein/plasminogen plate assay revealed that plasmid construct with the signal peptide of SK was superior to that with the signal peptide of sprT in SK production. The maximum productivity of SK was calculated as less than 0.25 unit/ml of the culture broth, which was similar level to those from other expression systems hiring ermE and tipA promoters in the same host. When the skc gene was expressed in E. coli BL21(DE3)pLys under control of T7 promoter, relatively large amount of SK was expressed in soluble form without hydrolyzed protein. The SK activity produced by E. coli/pET28a-T7pSKm was more than 2 units/ml of culture even though about half of the expressed protein formed inactive inclusion body.
从唾液链球菌（Streptococcus eouisimilis）ATCC9542 中克隆了编码链激酶（SK）的 skc 基因，其分子量约为 47.4 kDa，并在变铅青链霉菌（Streptomyces lividans）TK24 和大肠杆菌中使用各种强启动子进行了异源过表达。当在变铅青链霉菌 TK24 宿主中使用 sprT 启动子时，检测到与 47.4 kDa 相对应的 SK 蛋白有较小的水解蛋白（44 kDa），这表明如在其他表达系统中报道的那样发生了翻译后水解。酪蛋白/纤溶酶原平板测定表明，带有 SK 信号肽的质粒构建体在 SK 生产方面优于带有 sprT 信号肽的质粒构建体。SK 的最大产率计算为低于 0.25 单位/ml 培养液，与在相同宿主中使用 ermE 和 tipA 启动子的其他表达系统相当。当 skc 基因在大肠杆菌 BL21(DE3)pLys 中受 T7 启动子控制表达时，大量 SK 以可溶性形式表达，没有水解蛋白。即使约一半表达的蛋白形成无活性包涵体，大肠杆菌/pET28a-T7pSKm 产生的 SK 活性仍超过 2 单位/ml 培养物。
