title: Expression and characterization of human tyrosinase from a bacterial expression system.
abstract: To carry out biochemical characterizations of human tyrosinase and to provide an unlimited source of the enzyme for further study, an expression plasmid, pHis-Tyrosinase, which contains the entire coding sequence except the signal sequence of a human tyrosinase was constructed and expressed in Escherichia coli. The expressed enzyme was simply purified by an immobilized metal affinity chromatography. The recombinant enzyme had the same electrophoretic mobility as the native enzyme from human melanoma cell and cross-reacted with the polyclonal antibody raised against the native enzyme. The recombinant enzyme retained its catalytic function with both hydroxylating and oxidative activities. Km values for L-tyrosine and L-3,4-dihydroxy-phenylalanine of the recombinant enzyme were 0.17 and 0.36 mM, respectively. The activity of the recombinant enzyme was optimal at pH 7.5. Glutathione notably inhibited the enzymatic activity. This work is a further enzymatic characterization of human tyrosinase.
authors.affiliation: Department of Chemistry, College of Natural Science, Chung-Ang University, Seoul, South Korea. khkong@cau.ac.kr
authors.forename: K H
authors.initials: KH
authors.lastname: Kong
authors.forename: S Y
authors.initials: SY
authors.lastname: Park
authors.forename: M P
authors.initials: MP
authors.lastname: Hong
authors.forename: S H
authors.initials: SH
authors.lastname: Cho
mesh_terms: D004295:Dihydroxyphenylalanine / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D006031:Glycosylation; D006639:Histidine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D014442:Monophenol Monooxygenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D010455:Peptides; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D004295:Dihydroxyphenylalanine; D014442:Monophenol Monooxygenase