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Cell Journal، جلد ۱۵، شماره Suppl ۱، صفحات ۷۰-۷۰
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عنوان انگلیسی Ps-115: Comparative Studies of Native Human Tyrosinase and Its Two Mutational Variants in HEK-293 Cells
چکیده انگلیسی مقاله Objective: Pigmentation is one of the most obvious phenotypical characteristics in the natural world. Of the pigments, melanin is one of the most widely distributed and is found in bacteria, fungi, plants and animals. Melanins are heterogeneous polyphenol-like biopolymers with a complex structure and color varying from yellow to black. In the melanin formation pathway, tyrosinase is the rate-limiting enzyme that catalyzes tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and oxidizes DOPA to dopaquinone. Tyrosinases belong to type 3 copper proteins. The binuclear active site of tyrosinase consists of two copper ions, coordinated each by three highly conserved histidines. It has been shown that melanin biosynthesis pathway can be feasibly induced using recombinant tyrosinase. Our main aim to evaluate the activity of native tyrosinase and its two mutational variants in HEK-293 cells. Materials and Methods: Two mutants of human tyrosinase enzyme have been previously constructed in our lab anticipated to possess higher catalytic activity in HEK- 293 cells in comparison with the native protein. Native enzyme and the mutational variants have already been cloned in pET-28b(+) for evaluation of protein expression in Escherichia coli BL21. In this approach, coding sequences of native and mutant proteins have been subcloned into pcDNA3.1(+) expression vector followed by subsequent transfection of HEK-293 cells with three constructed pcDNA3.1(+)-hTyr vectors.Results: Digestion and sequencing results of constructed vectors all together showing that fragments have been cloned correctly. transfection's positive control also confirms transformation accuracy. Activity studies of native tyrosinase and two mutational variants have been performed and will be presented in congress. Conclusion: Human native tyrosinase and two mutational variants have been subcloned from a prokaryotic vector pET-28b(+) into pcDNA3.1+ for activity evaluation in eukaryotic cells.
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نشانی اینترنتی http://celljournal.org/journal/article/abstract/919
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