title: The dodecameric vanadium-dependent haloperoxidase from the marine algae Corallina officinalis: cloning, expression, and refolding of the recombinant enzyme.
abstract: The dodecameric vanadium-dependent bromoperoxidase from Corallina officinalis has been cloned and over-expressed in Escherichia coli. However, the enzyme was found to be predominantly in the form of inclusion bodies. This protein presents a challenging target for refolding, both due to the size (768kDa) and quaternary structure (12x64kDa). Successful refolding conditions have been established which result in an increase in the final yield of active bromoperoxidase from 0.5mg to 40mg per litre of culture. The refolded protein has been characterised and compared to the native enzyme and was shown to be stable at temperatures of 80 degrees C, over a pH range 5.5-10 and in organic solvents such as ethanol, acetonitrile, methanol, and acetone. The novel refolding approach reported in this paper opens up the full potential of this versatile enzyme for use in large scale biotransformation studies.
authors.affiliation: The Henry Wellcome Building for Biocatalysis, School of Biosciences, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: E E
authors.initials: EE
authors.lastname: Coupe
authors.forename: M G
authors.initials: MG
authors.lastname: Smyth
authors.forename: A P
authors.initials: AP
authors.lastname: Fosberry
authors.forename: R M
authors.initials: RM
authors.lastname: Hall
authors.forename: J A
authors.initials: JA
authors.lastname: Littlechild
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004338:Drug Combinations; D004926:Escherichia coli / Q000235:genetics; D056890:Eukaryota / Q000201:enzymology*; D015870:Gene Expression; D007453:Iodide Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008386:Marine Biology; D008969:Molecular Sequence Data; D009821:Oils; D010636:Phenols; D011108:Polymers; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D004338:Drug Combinations; D009821:Oils; C027222:P & S Liquid; D010636:Phenols; D011108:Polymers; D011994:Recombinant Proteins; C414928:vanadium iodoperoxidase; D007453:Iodide Peroxidase