Expression of the Trichoderma reesei tyrosinase 2 in Pichia pastoris: isotopic labeling and physicochemical characterization.
Trichoderma reesei tyrosinase TYR2 has been demonstrated to be able to oxidize various phenolic compounds and also peptide and protein bound tyrosine, and thus is of great interest for different biotechnological applications. In order to understand the reaction mechanism of the enzyme it would be essential to solve its three dimensional structure. Pichia pastoris is a suitable expression system for the production of recombinant enzymes for NMR studies and therefore we expressed TYR2 in this host. As a result of extensive optimization, the production yield of active histidine tagged tyrosinase purified from P. pastoris shake flask cultures was increased from 2.5 to 24 mg/L. Correct copper concentration in the growth medium was critical for the expression of this copper containing enzyme. Our analysis showed that TYR2 expressed in P. pastoris is post-translationally modified; the C-terminal domain of 153 amino acids of the protein is proteolytically cleaved off from the catalytic domain and the only potential N-glycosylation site is glycosylated. The activities of TYR2 expressed in P. pastoris and T. reesei on diphenolic L-dopa and monophenolic L-tyrosine were rather similar. The TYR2 expressed in P. pastoris showed the same physicochemical properties in CD and unfolding assays as the native TYR2 enzyme. Uniform isotopic (15)N-labeling of TYR2 was carried out with (15)NH(4)SO(4) in minimal medium to assess the suitability of the expression system for investigation by NMR spectroscopy.
Westerholm-Parvinen
Ann
A
VTT Technical Research Centre of Finland, PO Box 1000, Espoo FIN-02044 VTT, Finland.
Selinheimo
Emilia
E
Boer
Harry
H
Kalkkinen
Nisse
N
Mattinen
Maija
M
Saloheimo
Markku
M
D000595:Amino Acid Sequence; D003300:Copper / Q000032:analysis / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010843:Pichia / Q000235:genetics; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D014242:Trichoderma / Q000201:enzymology* / Q000254:growth & development
D003470:Culture Media; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D003300:Copper; D014442:Monophenol Monooxygenase