Implementing bacterial acid resistance into cell‐free protein synthesis for buffer‐free expression and screening of enzymes. Issue 12 (14th July 2015)

• Record Type: Journal Article
• Authors: Kim, Ho‐Cheol; Kim, Kwang‐Soo; Kang, Taek‐Jin; Choi, Jong Hyun; Song, Jae Jun; Choi, Yun Hee; Kim, Byung‐Gee; Kim, Dong‐Myung
• Is Part Of: Biotechnology and bioengineering. Volume 112:Issue 12(2015:Dec.)
• Page Start: 2630
• Page End: 2635
• Publication Date: 2015-07-14
• Subjects: cell‐free protein synthesis -- glutamate decarboxylase -- high‐throughput expression and screening -- α‐2, 3‐sialyltransferase -- pH homeostasis; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.25671
• Languages: English
• ISSNs: 0006-3592

Abstract

ABSTRACT: Cell‐free protein synthesis utilizes translational machinery isolated from the cells for in vitro expression of template genes. Because it produces proteins without gene cloning and cell cultivation steps, cell‐free protein synthesis can be used as a versatile platform for high‐throughput expression of enzyme libraries. Furthermore, the open nature of cell‐free protein synthesis allows direct integration of enzyme synthesis with subsequent screening steps. However, the presence of high concentration of chemical buffers in the conventional reaction mixture makes it difficult to streamline cell‐free protein synthesis with pH‐based assay of the synthesized enzymes. In this study, we have implemented an enzyme‐assisted bacterial acid resistance mechanism into an Escherichia coli (E.coli) extract‐based cell‐free protein synthesis system in place of chemical buffers. When deployed in the reaction mixture for cell‐free synthesis of enzymes, through proton‐consuming conversion of glutamate into γ‐aminobutyric acid (GABA), an engineered glutamate decarboxylase (GADβ) was able to maintain the pH of reaction mixture during enzyme synthesis. Because the reaction mixture becomes free of buffering capacity upon the depletion of glutamate, synthesized enzyme could be directly assayed without purification steps. The designed method was successfully applied to the screening of mutant library of sialyltransferase genes to identify mutants with improved enzymatic activity. Biotechnol. Bioeng. 2015;112: 2630–2635. © 2015 Wiley Periodicals, Inc. Abstract : The authors have implemented a bacterial acid resistance mechanism into an E. coli ‐based cell‐free protein synthesis system. Because the reaction mixture for cell‐free synthesis is virtually free of any chemical buffers, cell‐free synthesized enzymes can be directly screened by pH‐dependent assay methods. The designed method was successfully applied to the screening of mutant library of sialyltransferase genes to identify mutants with improved enzymatic activity.