title: Expression in non-melanogenic systems and purification of soluble variants of human tyrosinase.
abstract: Mammalian tyrosinases are key enzymes of melanin formation. Their native forms undergo complex maturation and sorting processes before being integrated into the melanosomal membrane, which greatly complicates their heterologous expression in other cell types. In the present work, we constructed several differently truncated, soluble variants of human tyrosinase and studied their properties after expression in HEK 293 cells. In addition, we prepared two affinity-tagged forms of the enzyme for expression in the yeast Kluyveromyces lactis and HEK cells, respectively. A Strep-tagged variant was secreted by K. lactis in excellent yields but found to be inactive, whereas a His-tagged variant secreted by HEK 293 cells in an active state could be purified from cell supernatants to near homogeneity. The resulting preparation consisted of an inactive, probably unglycosylated species of about 57 kDa and several glycosylated forms with masses between 63 and 75 kDa, as confirmed by activity staining, Western blotting and mass spectrometry.
authors.affiliation: Institute of Physiological Chemistry, Philipps University, Karl-von-Frisch-Strasse 1, D-35043 Marburg, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Cordes
authors.forename: Wei
authors.initials: W
authors.lastname: Sun
authors.forename: Rainer
authors.initials: R
authors.lastname: Wolber
authors.forename: Ludger
authors.initials: L
authors.lastname: Kolbe
authors.forename: Gerhard
authors.initials: G
authors.lastname: Klebe
authors.forename: Klaus-Heinrich
authors.initials: KH
authors.lastname: Röhm
mesh_terms: D002846:Chromatography, Affinity; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007716:Kluyveromyces / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008543:Melanins / Q000096:biosynthesis / Q000378:metabolism; D014442:Monophenol Monooxygenase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D014162:Transfection
chemical_list: D008543:Melanins; D014442:Monophenol Monooxygenase