title: Large-Scale Recombinant Expression and Purification of Human Tyrosinase Suitable for Structural Studies.
abstract: Human tyrosinase (TYR) is a glycoprotein that initiates the first two reactions in the melanin biosynthesis pathway. Mutations in its encoding gene cause Oculocutaneous Albinism type I (OCA1), the most severe form of albinism, which is a group of autosomal recessive disorders characterized by reduced or absent production of melanin in skin, hair and eyes. Despite extensive structural and characterization studies of its homologues in lower eukaryotic organisms, the catalytic mechanism of human TYR and the molecular basis of OCA1 are largely unknown. In this work, we have carried out a large-scale recombinant expression of TYR that has enabled us to obtain high yields of pure and active protein, required for crystallization trials and screening of skin whitening agents, which is highly demanded in the cosmetic industry. Addition of an N-terminal honeybee melittin signal peptide for secretion of the produced protein into the (protein-free) medium, as well as a cleavable His-tag at the C-terminus, was crucial for increasing the yield of pure protein. We have successfully crystallized two TYR variants, in both glycosylated and deglycosylated forms, showing preliminary X-ray diffraction patterns at 3.5 Å resolution. Hence, we have established an expression and purification protocol suitable for the crystal structure determination of human TYR, which will give unique atomic insight into the nature and conformation of the residues that shape the substrate binding pocket that will ultimately lead to efficient compound design.
authors.affiliation: Laboratory of Biophysical Chemistry, University of Groningen, Groningen, The Netherlands.|ESRF-The European Synchrotron, Grenoble, France.
authors.forename: Xuelei
authors.initials: X
authors.lastname: Lai
authors.affiliation: ESRF-The European Synchrotron, Grenoble, France.
authors.forename: Montserrat
authors.initials: M
authors.lastname: Soler-Lopez
authors.affiliation: Laboratory of Food Chemistry, Wageningen University, Wageningen, The Netherlands.
authors.forename: Harry J
authors.initials: HJ
authors.lastname: Wichers
authors.affiliation: Laboratory of Biophysical Chemistry, University of Groningen, Groningen, The Netherlands.
authors.forename: Bauke W
authors.identifier: http://orcid.org/0000-0001-9731-6586
authors.initials: BW
authors.lastname: Dijkstra
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002845:Chromatography; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D014442:Monophenol Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D055550:Protein Stability; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013696:Temperature; D014961:X-Ray Diffraction
chemical_list: D011994:Recombinant Proteins; D014442:Monophenol Monooxygenase