Cloning-independent expression and analysis of omega-transaminases by use of a cell-free protein synthesis system.
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.
Kwon
Yong-Chan
YC
Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, South Korea.
Lee
Kyung-Ho
KH
Kim
Ho-Cheol
HC
Han
Kyuboem
K
Seo
Joo-Hyun
JH
Kim
Byung-Gee
BG
Kim
Dong-Myung
DM
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*
D017931:DNA Primers; D000637:Transaminases