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.
authors: CICS - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6201-001 Covilhã, Portugal.
authors: A Q
authors: AQ
authors: Pedro
authors: M J
authors: MJ
authors: Bonifácio
authors: J A
authors: JA
authors: Queiroz
authors: C J
authors: CJ
authors: Maia
authors: L A
authors: LA
authors: Passarinha
mesh_terms: D061249:Batch Cell Culture Techniques; D058675:Brevibacillus; D002394:Catechol O-Methyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins; D002394:Catechol O-Methyltransferase