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A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltransferase

Paper ID Volume ID Publish Year Pages File Format Full-Text
23845 43477 2011 6 PDF Available
Title
A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltransferase
Abstract

Membrane proteins constitute 20–30% of all proteins encoded by the genome of various organisms. While large amounts of purified proteins are required for pharmaceutical and crystallization attempts, there is an unmet need for the development of novel heterologous membrane protein overexpression systems. Specifically, we tested the application of Brevibacillus choshinensis cells for the biosynthesis of human membrane bound catechol-O-methyltransferase (hMBCOMT). In terms of the upstream stage moderate to high expression was obtained for complex media formulation with a value near 45 nmol/h/mg for hMBCOMT specific activity achieved at 20 h culture with 37 °C and 250 rpm. Subsequently, the efficiency for reconstitution of hMBCOMT is markedly null in the presence of ionic detergents, such as sodium dodecyl sulphate (SDS). In general, for non-ionic and zwiterionic detergents, until a detergent critic micellar concentration (CMC) of 1.0 mM, hMBCOMT shows more biological activity at lower detergent concentrations while for detergent CMC higher than 1 mM, higher detergent concentrations seem to be ideal for hMBCOMT solubilization. Indeed, from the detergents tested, the non-ionic digitonin at 0.5% (w/v) appears to be the most suitable for hMBCOMT solubilization.

► Biosynthesis of human MBCOMT in Brevibacillus choshinensis cells for the first time. ► A high protein expression in complex media formulation with a value near of 45 nmol/h/mg. ► The recombinant enzyme is present in the membrane fraction and its recuperation is done easily by solubilization with a non-ionic detergent, digitonin or triton at 0.5% (w/v).

Keywords
B. choshinensis, Brevibacillus choshinensis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CMC, critic micellar concentration; COMT, catechol-O-methyltransferase; E. coli, Escherichia coli; hMBCOMT, human membrane-bound catechol-O-meth
First Page Preview
A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltransferase
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 156, Issue 2, 10 November 2011, Pages 141–146
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering