Cloning and expression of tyrosinase gene from Pseudomonas maltophilia in E. coli

The enzyme tyrosinase, encoded by tyrosinase gene (mel), is responsible for melanin formation. In a shotgun cloning experiment, a SalI-digested DNA fragment coding for tyrosinase was cloned from Pseudomonas maltophilia DNA into plasmid vector (pUC18) to generate the hybrid plasmid (pWSY). The recombinant plasmid imparted the ability of melanin synthesis to an E. coli host (HB010). The foreign DNA fragment (0.7kb) possessed no recognition sites for BamHI, HindIII, EcoRI or BclI. Hybridization studies confirmed that the small fragment cloned in pWSY was from P. maltophilia DNA. Nucleotide sequence analysis identified an ORF of 504nt coding tyrosinase. SDS-PAGE analysis also revealed an additional protein of 18kDa, which was equal to the putative tyrosinase according to the size of mel fragment, was expressed in the E. coli recombinant carrying the plasmid pWSY.