Oh In-Seok, Kim Dong-Myung, Kim Tae-Wan, Park Chang-Gil, Choi Cha-Yong
School of Chemical and Biological Engineering, College of Engineering, Seoul National University, Korea.
Biotechnol Prog. 2006 Jul-Aug;22(4):1225-8. doi: 10.1021/bp060051l.
We developed a novel method of producing proteins containing multiple disulfide bonds in a cell-free protein synthesis system. To provide an optimized redox potential during the synthesis of truncated plasminogen activator (rPA), we pretreated the E. coli S30 extract with an excess amount of oxidized glutathione based on the anticipation that the reducing potential of the S30 extract would be exhausted through the reduction of the oxidized glutathione molecules. As expected, it was found that the reducing activity of the S30 extract was remarkably decreased through the pretreatment, and active rPA was produced when the pretreated S30 extract was used after removing the residual glutathione molecules. In particular, compared to the method involving the iodoacetamide treatment of S30 extract, the present protocol was effective in producing active rPA during the batch reaction of cell-free protein synthesis.
我们开发了一种在无细胞蛋白质合成系统中生产含有多个二硫键蛋白质的新方法。为了在截短型纤溶酶原激活剂（rPA）合成过程中提供优化的氧化还原电位，我们基于S30提取物的还原电位会因氧化型谷胱甘肽分子的还原而耗尽的预期，用过量的氧化型谷胱甘肽预处理大肠杆菌S30提取物。正如预期的那样，发现通过预处理S30提取物的还原活性显著降低，并且在去除残留的谷胱甘肽分子后使用预处理的S30提取物时产生了活性rPA。特别是，与对S30提取物进行碘乙酰胺处理的方法相比，本方案在无细胞蛋白质合成的分批反应过程中有效产生活性rPA。
