High activity expression of d-amino acid oxidase in Escherichia coli by the protein expression rate optimization

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• 作者：Jixian Hou ; Yangqiu Liu ; Qiang Li ; ^{liqiang@tsinghua.edu.cn} ; Jichu Yang
• 关键词：Protein expression rate ; Ribosome binding site region ; d-amino acid oxidase
• 刊名：Protein Expression and Purification
• 出版年：2013
• 出版时间：March, 2013
• 年：2013
• 卷：88
• 期：1
• 页码：120-126
• 全文大小：482 K

文摘

Two categories of expression systems with different promoters and substitutive ribosome binding site region (RBS) were constructed in order to improve the soluble expression of d-amino acid oxidase (DAAO) basing on the hypothesis that the optimal promoter and suitable RBS would provide the recombinant expression system with better matched expression rate, which served as key factor to the heterogenous synthesis of soluble protein. The results showed that with rational promoter recombination and delicate RBS substitution, significant increase of DAAO activity (20-fold) was obtained for strain JM105/pGEMKT-Tac-R-DAAO over the previously constructed strain BL21(DE3)/pET-DAAO. Furthermore, similar optimization strategy proved feasible in the active expression of other enzymes such as glutaryl-7-aminocephalosporanic acid acylase (GCA) and N-Carbamyl-d-amino acid amidohydrolase (d-Case).