title: Expression of recombinant mature human tyrosinase from Escherichia coli and exhibition of its activity without phosphorylation or glycosylation.
abstract: A cDNA encoding mature human tyrosinase was cloned into pET-23a(+) and transformed into E. coli BL21(DE3). Three major recombinant proteins, mature human tyrosinase (RHT₂₀₋₅₃₁), N-terminal truncated human tyrosinase (RHT₁₆₈₋₅₃₁), and β-lactamase, were overexpressed as inclusion bodies in E. coli after 12 h of induction with 1.0 mM isopropyl-β-D-thiogalactopyranoside at 37 °C. After sonication and centrifugation, the inclusion body was harvested, solubilized, dialyzed, and refolded into the active form with monophenolase and diphenolase activities. It was purified to homogeneity by DEAE-Sepharose FF and Sephadex G-75. The molecular mass and N-terminal sequence were 57.0 kDa and GHFPRAC, respectively, and corresponded to those of mature human tyrosinase. The RHT was active in a broad range of temperature and pH, and with optimum activity at 70 °C and pH 8.5.
authors.affiliation: Department of Cosmetic Science, Providence University, Salu, Taichung, Taiwan 43301.
authors.forename: Gen-Hung
authors.initials: GH
authors.lastname: Chen
authors.forename: Wei-Ming
authors.initials: WM
authors.lastname: Chen
authors.forename: Ya-Chi
authors.initials: YC
authors.lastname: Huang
authors.forename: Shann-Tzong
authors.initials: ST
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D045744:Cell Line, Tumor; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014442:Monophenol Monooxygenase