Tyrosinase (EC 184.108.40.206) 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.
|Conference||3rd European Federation of Biotechnology Conference |
|Period||13/06/07 → 16/06/07|