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Providing an Oxidizing Environment for the Cell‐Free Expression of Disulfide‐Containing Proteins by Exhausting the Reducing Activity of Escherichia coli S30 Extract
Author(s) -
Oh InSeok,
Kim DongMyung,
Kim TaeWan,
Park ChangGil,
Choi ChaYong
Publication year - 2006
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp060051l
Subject(s) - iodoacetamide , glutathione , oxidizing agent , reducing agent , chemistry , escherichia coli , biochemistry , redox , disulfide bond , activator (genetics) , cell free protein synthesis , cysteine , combinatorial chemistry , protein biosynthesis , organic chemistry , enzyme , gene
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.