Cloning-Independent Expression and Analysis of ω-Transaminases by Use of a Cell-Free Protein Synthesis System

• Published in: Applied and Environmental Microbiology 2010-09, Vol.76 (18), p.6295-6298
• Main Authors: Kwon, Yong-Chan, Lee, Kyung-Ho, Kim, Ho-Cheol, Han, Kyuboem, Seo, Joo-Hyun, Kim, Byung-Gee, Kim, Dong-Myung
• Format: Article
• Language: English
• Subjects: amino acid sequences; bacteria; Biological and medical sciences; Biotechnology; Cell-Free System; Chromatography, High Pressure Liquid; Colorimetry; DNA Primers - genetics; enzyme activity; Fundamental and applied biological sciences. Psychology; Gene Expression; genes; High-Throughput Screening Assays - methods; Microbiology; Nucleic Acid Amplification Techniques; nucleotide sequences; Polymerase Chain Reaction; prediction; protein synthesis; screening; Substrate Specificity; transaminases; Transaminases - genetics
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.00029-10

Herewith we report the expression and screening of microbial enzymes without involving cloning procedures. Computationally predicted putative ω-transaminase (ω-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 ω-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.