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.
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.