Expression and characterization of human tyrosinase from a bacterial expression system

To carry out biochemical characterizations of human tyrosinase and to provi de an unlimited source of the enzyme for further study, an expression plasm id, pHis-Tyrosinase, which contains the entire coding sequence except the s ignal sequence of a human tyrosinase was constructed and expressed in Esche richia coli. The expressed enzyme was simply purified by an immobilized met al affinity chromatography. The recombinant enzyme had the same electrophor etic mobility as the native enzyme from human melanoma cell and cross-react ed with the polyclonal antibody raised against the native enzyme. The recom binant enzyme retained its catalytic function with both hydroxylating and o xidative activities. K-m values for L-tyrosine and L-3,4-dihydroxy-phenylal anine of the recombinant enzyme were 0.17 and 0.36 mM, respectively. The ac tivity of the recombinant enzyme was optimal at pH 7.5. Glutathione notably Inhibited the enzymatic activity. This work is a further enzymatic charact erization of human tyrosinase. (C) 2000 Elsevier Science Inc. All rights re served.