| Summary: | 碩士 === 靜宜大學 === 化粧品科學系 === 107 === Human tyrosinase, a kind of membrane protein, integrated into melanosome membrane through its C- terminal transmembrane domain. The hydrophobic transmembrane domain will cause protein aggregation during expression and/or renaturation without the membrane system. In this study, the cDNA encoding recombinant human tyrosinase (RHT) excluded transmembrane domain was therefore cloned into pET23a(+) vector using T7 as promoter and following transformed into E. coli BL21(DE3) expression host. After 4 hours shaking cultivation at 37oC, high quantity of the RHT was expressed as soluble form in the cytosol. The N-terminal sequence was GHFPRAC corresponded to mature human tyrosinase. There results suggested that deletion of C- terminal transmembrane domain could exactly avoid aggregation during expression and express in soluble form in the cytosol of E. coli. After refolding of the inclusion bodies, the optimum pH value is pH 9.0, the same as that of the recombinant full-length human tyrosinase, but the optimum temperature is 80℃, higher than that of the recombinant full-length human tyrosinase by 10℃. On the other hand, the cDNA encoding recombinant human tyrosinase (RHT) excluded c-terminal transmembrane domain was cloned into pPIC9 vector using AOX1 as promoter. After transformation of the RHT-pPIC9 into Pichia pastoris GS115 expression host, the RHT-pPIC9 vector was integrated into the AOX1 promoter locus of Pichia pastoris GS115 chromosome. After 5 days shaking cultivation at 30 oC, high quantity of the RHT was expressed and secreted into the broth using α-factor preprosequence. The N-terminal sequence was GHFPRAC corresponded to mature human tyrosinase.
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