Expression of the Trichoderma reesei Tyrosinase 2 in Pichia pastoris: Isotopic labeling and physicochemical characterization

Ann Westerholm-Parvinen, Emilia Selinheimo, Harry Boer, Nisse Kalkkinen, Maija Mattinen, Markku Saloheimo (Corresponding Author)

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Abstract

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 15N-labeling of TYR2 was carried out with 15NH4SO4 in minimal medium to assess the suitability of the expression system for investigation by NMR spectroscopy.
Original languageEnglish
Pages (from-to)147-158
JournalProtein Expression and Purification
Volume55
Issue number1
DOIs
Publication statusPublished - 2007
MoE publication typeA1 Journal article-refereed

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Keywords

  • Tyrosinase
  • Cu enzymes
  • Pichia pastoris
  • heterologous expression
  • NMR

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