Heterologous expression of isotopically labelled Trichoderma reesei tyrosinase 2 in Pichia pastoris

Tyrosinase (EC 1.14.18.1) is a copper-containing oxidase that is widely distributed in mammals, invertebrates, plants and microorganisms. In mammals the enzyme is essential for the formation of melanin pigments, whereas tyrosinases in fruit and vegetables are related to the browning reaction that occurs upon bruising and long term storage. Tyrosinase is of great interest for many applications in the field of medicine, biotechnology and food engineering. It is a promising target enzyme for prodrug activations in melanomas and in biotechnological applications including crosslinking of protein matrices. It is of great importance to find ligands and inhibitors for tyrosinase. Structural studies and screening for ligands and inhibitors can be carried out using NMR spectroscopy with isotopically labeled tyrosinase. Therefore, we cloned a novel tyrosinase from Trichoderma reesei and expressed it heterologously in the methylotrophic yeast Pichia pastoris. A novel tyrosinase, tyrosinase 2 (TYR2), was cloned from Trichoderma reesei. The cDNA sequence was expressed under the control of the AOX1 promoter in the Pichia pastoris X-33 strain. The Saccharomyces cerevisiae alpha-MF prepro sequence was used for secretion and an N-terminal His6-tag was fused to the tyrosinase to facilitate the detection and purification of the recombinant protein. Heterologous expression was carried out in shake flask cultivations and the enzymatic activity was measured directly on the culture medium, using L-Dopa as a substrate. Extensive optimisation of the expression in shake flasks was carried out as the stable isotope labels are costly. Different temperatures, different CuSO4 and NH4SO4 concentrations and different shake flasks were tested. The expression level of recombinant TYR2 was increased tenfold as a result of the optimisation. Metabolic 15N-labeling of TYR2 was carried out with 15NH4SO4 in minimal medium to assess its suitability for investigations by NMR spectroscopy. Initial 3D heteronuclear 1H-15N HSQC NMR spectrum of TYR2 showed signals with chemical shifts typical of folded proteins. The Trichoderma reesei tyrosinase 2 was successfully expressed and uniformly 15N labeled in the yeast Pichia pastoris. This methylotrophic yeast is a suitable expression system for the production of recombinant proteins for NMR studies as it is cost-effective and possesses the ability to perform many of the posttranslational modifications of higher eukaryotes.