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Cloning-independent expression and screening of enzymes using cell-free protein synthesis systems.

Authors :
Kwon YC
Song JK
Kim DM
Source :
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2014; Vol. 1118, pp. 97-108.
Publication Year :
2014

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. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function, in order to accelerate the discovery of novel enzymes. The proposed strategy can also serve as a viable option for the rapid and precise tuning of enzyme molecules, not only for analytical purposes, but also for industrial applications. This is accomplished via large-scale production using microbial cells transformed with variant genes selected from the cell-free expression screening.

Details

Language :
English
ISSN :
1940-6029
Volume :
1118
Database :
MEDLINE
Journal :
Methods in molecular biology (Clifton, N.J.)
Publication Type :
Academic Journal
Accession number :
24395411
Full Text :
https://doi.org/10.1007/978-1-62703-782-2_6