A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltransferase |
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Authors: | Pedro A Q Bonifácio M J Queiroz J A Maia C J Passarinha L A |
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Institution: | a CICS - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6201-001 Covilhã, Portugal b Bial - Departamento de Investigação e Desenvolvimento, 4745-457 São Mamede do Coronado, Portugal |
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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. |
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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-methyltransferase MBCOMT membrane-bound catechol-O-methyltransferase SAM l-methionine" target="_blank">S-adenosyl-l-methionine SCOMT catechol-O-methyltransferase soluble isoform SDS sodium dodecyl sulphate |
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