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Title 

Implementing bacterial acid resistance into cell-free protein synthesis for buffer-free expression and screening of enzymes

Authors 

H C KimK S KimT J KangJong Hyun ChoiJae Jun SongY H ChoiB G KimD M Kim

Publisher 

Wiley-Blackwell

Issue Date 

2015

Citation 

Biotechnology and Bioengineering, vol. 112, no. 12, pp. 2630-2635

Keywords 

Cell-free protein synthesisGlutamate decarboxylaseHigh-throughput expression and screeningPH homeostasisα-2,3-sialyltransferase

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.

ISSN 

0006-3592

Link 

http://dx.doi.org/10.1002/bit.25671

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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