Construction and characterization of Pichia pastoris strains for labeling aromatic amino acids in recombinant proteins

Mei M. Whittaker, James Whittaker

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Strains of the methylotrophic yeast Pichia pastoris auxotrophic for the aromatic amino acids (tyrosine, phenylalanine, and tryptophan) have been constructed by targeted gene disruption for protein labeling applications. Three strains, with defects in ARO1 (coding for a homolog of the arom pentafunctional enzyme), ARO7 (coding for chorismate mutase), and TYR1 (coding for prephenate dehydrogenase), have been engineered in a P. pastoris ura3Δ1 parent strain using standard methods. The nutritional requirements of these auxotrophic strains have been characterized and their utility as expression hosts for labeling recombinant proteins has been demonstrated. All three strains show a surprising sensitivity to rich culture medium and must be grown in supplemented minimal medium. The tyr1::URA3 strain in particular is strongly inhibited by tryptophan, and to a lesser extent by phenylalanine, leucine, and isoleucine. Highly efficient incorporation of exogenously supplied amino acids by these three auxotroph strains has been demonstrated using recombinant galactose oxidase. Stereochemically pure l-amino acids and racemic d,l-mixtures serve nearly equally well to support protein expression and labeling. These strains allow efficient labeling of aromatic amino acids in recombinant proteins, supporting NMR structural biology and a wide range of other biophysical studies.

Original languageEnglish (US)
Pages (from-to)266-274
Number of pages9
JournalProtein Expression and Purification
Volume41
Issue number2
DOIs
StatePublished - Jun 2005

Fingerprint

Aromatic Amino Acids
Pichia
Phenylalanine
Recombinant Proteins
Tryptophan
Labeling
Prephenate Dehydrogenase
Chorismate Mutase
Galactose Oxidase
Amino Acids
Nutritional Requirements
Isoleucine
Leucine
Tyrosine
Culture Media
Proteins
Yeasts
Yeast
Genes
Nuclear magnetic resonance

Keywords

  • Aromatic amino acids
  • Auxotroph
  • Galactose oxidase
  • Pichia pastoris
  • Strain construction

ASJC Scopus subject areas

  • Biochemistry

Cite this

Construction and characterization of Pichia pastoris strains for labeling aromatic amino acids in recombinant proteins. / Whittaker, Mei M.; Whittaker, James.

In: Protein Expression and Purification, Vol. 41, No. 2, 06.2005, p. 266-274.

Research output: Contribution to journalArticle

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