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Methods in Molecular Biology
Vol. 1118, No: xx, 2014, Pages:
97-108

Cloning-Independent Expression and Screening of Enzymes Using Cell-Free Protein Synthesis Systems

Yong-Chan Kwon, Jae-Kwang Song, Dong-Myung Kim

Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon, South Korea.

Abstract

We present a strategy for expression and screening of microbial enzymes without involving cloning procedures. Libraries of putative ω-transaminases (ω-TA) and mutated Candida antarctica lipase B (CalB) are PCR-amplified from bacterial colonies and directly expressed in an Escherichia coli-based cell-free protein synthesis system. The open nature of cell-free protein synthesis system also allows streamlined analysis of the enzymatic activity of the expressed enzymes, which greatly shortens the time required for enzyme screening.

Keywoards: Enzyme engineering; Combinatorial mutagenesis; Cell-free protein synthesis; Hot spots.


 
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