Expression, Purification and Activity Assay of the Recombinant Protein of Catechol-O-Methyltransferase from Chinese White Shrimp (&lt;i&gt;Fenneropenaeus chinensis&lt;/i&gt;)

Li Li

doi:10.3844/ajbbsp.2010.148.154

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.

Highlights

Catechol-O-Methyltransferase (COMT, E.C.2.1.1.6.) developed as adjuvant drugs in the treatment of is one of O-methyltransferases that catalyse the Parkinson's disease (Schrag, 2005)
Activity assay of recombinant Fc-COMT using High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS): Using Dihydroxybenzoic Acid (DHBAc) as substrate, the enzymatic reaction products of Vanillic Acid (VA) and Isovanillic Acid (IVA) were detected by HPLC-MS according to methods previously described (Li et al, 2004; Reenila and Rauhala, 2009; Reenila et al, 1995)
Expression and purification of recombinant FcCOMT: When sequencing analysis confirmed that the DNA sequence and protein sequence of pET30a(+)/FcCOMT were correct, the recombinant plasmid was transformed into E. coli cells and recombinant Fc-COMT was induced by isopropyl β-DThiogalactopyranoside (IPTG)

Summary

Introduction

Catechol-O-Methyltransferase (COMT, E.C.2.1.1.6.) developed as adjuvant drugs in the treatment of is one of O-methyltransferases that catalyse the Parkinson's disease (Schrag, 2005). COMT is formation of methoxylated products by transferring one involved in the studies of pharmacology and etiology for methyl group from S-adenosyl-L-methionine to the some diseases. No crustacean COMT, other catechol-type compounds including many catechol- except for Chinese white shrimp (F. chinensis) COMT containing xenobiotics and drugs (Mannisto et al, 1992; (Fc-COMT), has yet been reported. Researchers have achieved a lot in COMT studies expression pattern (Li et al, 2006). The sequence data including gene cloning (Bertocci et al, 1991), gene of the Fc-COMT gene has been submitted to the expressions In order to further analyze the enzymatic enzyme kinetics (Bonifacio et al, 2002) and enzyme activity of the Fc-COMT, this study was conducted

Methods

Results

Conclusion