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)

Research output: Contribution to journalArticleScientificpeer-review

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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

Fingerprint

Trichoderma
Pichia
Monophenol Monooxygenase
Enzymes
Tyrosine
Copper
Dihydroxyphenylalanine
Batch Cell Culture Techniques
Glycosylation
Histidine
Catalytic Domain
Proteins
Magnetic Resonance Spectroscopy
Amino Acids
Peptides
Growth

Keywords

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

Cite this

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title = "Expression of the Trichoderma reesei Tyrosinase 2 in Pichia pastoris: Isotopic labeling and physicochemical characterization",
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.",
keywords = "Tyrosinase, Cu enzymes, Pichia pastoris, heterologous expression, NMR",
author = "Ann Westerholm-Parvinen and Emilia Selinheimo and Harry Boer and Nisse Kalkkinen and Maija Mattinen and Markku Saloheimo",
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Expression of the Trichoderma reesei Tyrosinase 2 in Pichia pastoris: Isotopic labeling and physicochemical characterization. / Westerholm-Parvinen, Ann; Selinheimo, Emilia; Boer, Harry; Kalkkinen, Nisse; Mattinen, Maija; Saloheimo, Markku (Corresponding Author).

In: Protein Expression and Purification, Vol. 55, No. 1, 2007, p. 147-158.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Expression of the Trichoderma reesei Tyrosinase 2 in Pichia pastoris: Isotopic labeling and physicochemical characterization

AU - Westerholm-Parvinen, Ann

AU - Selinheimo, Emilia

AU - Boer, Harry

AU - Kalkkinen, Nisse

AU - Mattinen, Maija

AU - Saloheimo, Markku

PY - 2007

Y1 - 2007

N2 - 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.

AB - 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.

KW - Tyrosinase

KW - Cu enzymes

KW - Pichia pastoris

KW - heterologous expression

KW - NMR

U2 - 10.1016/j.pep.2007.04.014

DO - 10.1016/j.pep.2007.04.014

M3 - Article

VL - 55

SP - 147

EP - 158

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 1

ER -