title: Human tyrosinase produced in insect cells: a landmark for the screening of new drugs addressing its activity.
abstract: Human tyrosinase is the first enzyme of the multistep process of melanogenesis. It catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine and the following oxidation of o-diphenol to the corresponding quinone, L-dopaquinone. In spite of its biomedical relevance, its reactivity is far from being fully understood, mostly because of the lack of a suitable expression system. Indeed, until now, studies on substrates and inhibitors of tyrosinases have been performed in vitro almost exclusively using mushroom or bacterial enzymes. We report on the production of a recombinant human tyrosinase in insect cells (Sf9 line). Engineering the protein, improving cell culture conditions, and setting a suitable purification protocol optimized product yield. The obtained active enzyme was truthfully characterized with a number of substrate and inhibitor molecules. These results were compared to those gained from a parallel analysis of the bacterial (Streptomyces antibioticus) enzyme and those acquired from the literature for mushroom tyrosinase, showing that the reactivity of the human enzyme appears unique and pointing out the great bias introduced when using non-human tyrosinases to measure the inhibitory efficacy of new molecules. The described enzyme is therefore an indispensable paradigm in testing pharmaceutical or cosmetic agents addressing tyrosinase activity.
authors.affiliation: Department of Biology, University of Padua, Viale G. Colombo 3, 35121, Padua, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Fogal
authors.forename: Marcello
authors.initials: M
authors.lastname: Carotti
authors.forename: Laura
authors.initials: L
authors.lastname: Giaretta
authors.forename: Federico
authors.initials: F
authors.lastname: Lanciai
authors.forename: Leonardo
authors.initials: L
authors.lastname: Nogara
authors.forename: Luigi
authors.initials: L
authors.lastname: Bubacco
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Bergantino
mesh_terms: D000363:Agaricales / Q000201:enzymology; D000818:Animals; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D007313:Insecta / Q000378:metabolism*; D007700:Kinetics; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D061987:Sf9 Cells; D013302:Streptomyces / Q000201:enzymology; D014780:Viruses / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D011994:Recombinant Proteins; D014442:Monophenol Monooxygenase