title: Cloning-independent expression and analysis of omega-transaminases by use of a cell-free protein synthesis system.
abstract: Herewith we report the expression and screening of microbial enzymes without involving cloning procedures. Computationally predicted putative omega-transaminase (omega-TA) genes were PCR amplified from the bacterial colonies and expressed in a cell-free protein synthesis system for subsequent analysis of their enzymatic activity and substrate specificity. Through the cell-free expression analysis of the putative omega-TA genes, a number of enzyme-substrate pairs were identified in a matter of hours. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function to accelerate the discovery of novel enzymes.
authors.affiliation: Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, South Korea.
authors.forename: Yong-Chan
authors.initials: YC
authors.lastname: Kwon
authors.forename: Kyung-Ho
authors.initials: KH
authors.lastname: Lee
authors.forename: Ho-Cheol
authors.initials: HC
authors.lastname: Kim
authors.forename: Kyuboem
authors.initials: K
authors.lastname: Han
authors.forename: Joo-Hyun
authors.initials: JH
authors.lastname: Seo
authors.forename: Byung-Gee
authors.initials: BG
authors.lastname: Kim
authors.forename: Dong-Myung
authors.initials: DM
authors.lastname: Kim
mesh_terms: D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D057166:High-Throughput Screening Assays / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity; D000637:Transaminases / Q000235:genetics*
chemical_list: D017931:DNA Primers; D000637:Transaminases