Research Article Open Access

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

Dian-Xiang Li1, Xin-Jun Du2, Xiao-Fan Zhao2 and Jin-Xing Wang2
  • 1 University of Jinan, China
  • 2 Shandong University, China

Abstract

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.

American Journal of Biochemistry and Biotechnology
Volume 6 No. 3, 2010, 148-154

DOI: https://doi.org/10.3844/ajbbsp.2010.148.154

Submitted On: 13 August 2010 Published On: 30 September 2010

How to Cite: Li, D., Du, X., Zhao, X. & Wang, J. (2010). Expression, Purification and Activity Assay of the Recombinant Protein of Catechol-O-Methyltransferase from Chinese White Shrimp (Fenneropenaeus chinensis). American Journal of Biochemistry and Biotechnology, 6(3), 148-154. https://doi.org/10.3844/ajbbsp.2010.148.154

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Keywords

  • Fenneropenaeus chinensis
  • recombinant Fc-COMT
  • VA
  • HPLC-MS