American Journal of Biochemistry and Biotechnology

Expression, Purification and Activity Assay of the Recombinant Protein of Catechol-O-Methyltransferase from Chinese White Shrimp (Fenneropenaeus chinensis)

Dian-Xiang Li, Xin-Jun Du, Xiao-Fan Zhao and Jin-Xing Wang

DOI : 10.3844/ajbbsp.2010.148.154

American Journal of Biochemistry and Biotechnology

Volume 6, Issue 3

Pages 148-154


Problem statement: We have previously cloned a gene of Chinese white shrimp Catechol O-Methyltransferase (designated Fc-COMT) and characterized the gene expression pattern. In this study, expression and purification as well as activity assay of the recombinant Fc-COMT was further conducted. Approach: Using pET-30a (+) as a prokaryotic expression vector, the recombinant Fc- COMT was expressed in the supernatant of Escherichia coli lysate and easily purified by His-Bind resin chromatography. SDS-PAGE analysis showed that the molecular mass of recombinant Fc-COMT was approximately 30,000 Da, in good agreement with the software-predicted molecular weight. The enzymatic activity of recombinant Fc-COMT was tested using Dihydroxybenzoic Acid (DHBAc) as a substrate. Results: The methyl products of DHBAc, Vanillic Acid (VA) and Isovanillic Acid (IVA), were detected in the enzymatic reaction mixture with recombinant Fc-COMT by High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS). Conclusion: The recombinant Fc-COMT has catalytic activity of transferring methyl group from S-Adenosyl-L-Methionine (SAM) to the 3' hydroxyl or 4' hydroxyl group of benzyl ring of DHBAc.


© 2010 Dian-Xiang Li, Xin-Jun Du, Xiao-Fan Zhao and Jin-Xing Wang. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.