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Nicole Joanne Kus, Monika B Dolinska, Yuri V Sergeev; Purification and biochemical characterization of full-length human tyrosinase. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1723.
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The human tyrosinase (hTyr) is a Type I membrane bound glycoenzyme located in the melanosome. Tyrosinase is responsible for the production of melanin, catalyzing the initial and rate-limiting steps in melanogenesis, namely the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (L-dopa) and subsequent oxidation of o-diphenol to L-dopaquinone. About ~300 mutations in the hTyr gene are associated with oculocutaneous albinism (OCA-1), an autosomal recessive disorder. Previously, the intra-melanosomal domain of human tyrosinase (hTyrCtr) has been expressed, purified and characterized in our group. Here, we purified and characterized the hTyr and compared this protein with properties of the hTyrCtr.
Both hTyr and hTyrCtr were produced in T. ni larvae. The membrane-bound hTyr was solubilized in the presence of 1.0% Triton X-100. HisTrap affinity and size exclusion chromatography were used in the purification process, where all buffers contained 0.1% Triton X-100. The obtained fractions were treated with Proteinase K (100 mg/mL) followed by Superose 12 10/300 gel filtration. Production of the hTyr obtained a yield of >0.1 mg per 10 g of larval biomass. hTyrCtr was purified as a regular soluble protein. Molecular masses were determined from SEC profiles using Bio-Rad protein standards. Michaelis-Menten kinetics measured using a Spectramax i3 plate reader.
Western Blot and SDS-Page showed the hTyr polypeptide molecular weight from 67 to 98 kDa. The polydispersion caused a range of molecular mass attributed to the variability of protein glycosylation. The molecular mass of hTyr was obtained from a single peak in the gel filtration chromatogram, approximately 74,131 Da, in comparison to hTyrCtr, which had a molecular mass of 56,742 Da. Activity of the protein was measured in the presence of detergent. An active, approximately 10% glycosylated, hTyr protein was obtained which had a Km of 0.769±0.267 mM, similar to hTyrCtr (0.77±0.335 mM).
Full-length human tyrosinase was successfully over expressed in T. ni larvae, solubilized, and purified in the presence of Triton X-100. Similarity of the Michaelis constant suggests similar ligand binding affinities for hTyr and hTyrCtr. Purified tyrosinase is critical for the search of suitable activators of mutant variants in treatment of genetic disorders, such as oculocutaneous albinism (OCA-1), and dermatological disorders, like hyperpigmentation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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