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KMID : 1094720070120050574
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Biotechnology and Bioprocess Engineering
2007 Volume.12 No. 5 p.574 ~ p.578
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Use ofl -buthionine sulfoximine for the efficient expression of disulfide-containing proteins in cell-free extracts of Escherichia coli
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Oh In-Seok
Kim Tae-Wan
Ahn Jin-Ho
Keum Jung-Won
Choi Cha-Yong
Kim Dong-Myung
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Abstract
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We have developed a technique to improve the formation of correct disulfide bonds within cell-free synthesized proteins. Via the use of a metabolic inhibitor of glutamate-cysteine ligase, the accumulation of glutathione was effectively prevented in cell-free extracts, thereby enabling the stable maintenance of redox potential for extended reaction periods. As a result, in a reaction in which a model protein contatining 9 disulfide bonds was synthesized under cell-free conditions, the final amount of active protein products was increased by 50%. The method presented in this study will provide a rapid and robust route to the high-throughput expression and screening of proteins which require multiple disulfide bonds for their activity.
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KEYWORD
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cell-free protein synthesis system, disulfide bond, glutathione, l-buthionine sulfoximine, recombinant plasminogen activator, sulfhydryl redox potential, S30 extract
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