Cloning Of Tyrosinase Gene And The Expression Of Its Wild-type As Well As Variants In Escherichia.coli

Tyrosinase, a copper-containing enzyme,.plays a key role in melanin metabolism and catecholamine synthesis. Tyrosinase was widely investigated for a long time. However, limited information on the relation of biocharacter and function of tyrosinase was obtained because of the limited acquisition of tyrosinase. Here we reported the cloning of the gene from murine tyrosinase, the expression of wild tyrosinase and variants in E.coli. The results were showed in following:Gene cloning and expression plasmid construction: the cDNA encoding murine tyrosinase was amplified from the total RNA by RT-PCR. By using the technology of DNA recombination, the cDNA was reconstructed and ligated into expression vector pET-22b(+) with T7 promoter and pGEX-2T with tac promoter, successfully constructed pET-TYR(wild type) and variant pET-Tat-TYR (Twin Arginine Transport variant type) and pGEX-TYR (GST fusion type with the lack of transmembrane region).Expression of wild tyrosinase: the pET-TYR was transformed into BL21(DE3), but no tyrosinase expressed was discovered. When the pET-TYR was transformed into Rosetta(DE3) the target protein was expressed and identified by amino acid sequencing.Expression in Twin Arginine Transport system: pET-Tat-TYR was transformed into Rosetta (DE3). Then Rosetta (DE3) was induced with IPTG (1.0mmol/L) for 4 hours at 37℃, and the Tat-tyrosinase was successfully expressed.Expression of GST fusion system: pGEX-TYR was transformed into BL21 (DE3), the protein GST-tyrosinase can also be expressed, but the expressed GST-tyrosinase is in the form of inclusion bodies devoid of activity.The optimization of the induce conditions: induce temperature, time and concentrations of IPTG were mainly considered. We found that soluble protein can be expressed by using pGEX-TYR, which was induced at 30℃ (or 16℃) over night. But no activity was discovered. These results indicated that: the active recombinant tyrosinase was hard to be produced by E.coli expression system. Possibly, it is due to failure of glycosylation in E.coli. These findings establish a certain foundation for the expression of tyrosinase and the analysis of structure and function in the future.