Improved strategy for isoleucine 1H/13C methyl labeling in Pichia pastoris

Rustam Ali, Lindsay D. Clark, Jacob A. Zahm, Andrew Lemoff, Karthik Ramesh, Daniel M. Rosenbaum, Michael K. Rosen

Research output: Contribution to journalArticle

Abstract

Site specific methyl labeling combined with methyl TROSY offers a powerful NMR approach to study structure and dynamics of proteins and protein complexes of high molecular weight. Robust and cost-effective methods have been developed for site specific protein 1H/13C methyl labeling in an otherwise deuterated background in bacteria. However, bacterial systems are not suitable for expression and isotope labeling of many eukaryotic and membrane proteins. The yeast Pichia pastoris (P. pastoris) is a commonly used host for expression of eukaryotic proteins, and site-specific methyl labeling of perdeuterated eukaryotic proteins has recently been achieved with this system. However, the practical utility of methyl labeling and deuteration in P. pastoris is limited by high costs. Here, we describe an improved method for 1H/13C-labeling of the δ-methyl group of isoleucine residues in a perdeuterated background, which reduces the cost by ≥ 50% without compromising the efficiency of isotope enrichment. We have successfully implemented this method to label actin and a G-protein coupled receptor. Our approach will facilitate studies of the structure and dynamics of eukaryotic proteins by NMR spectroscopy.

Original languageEnglish (US)
JournalJournal of Biomolecular NMR
DOIs
StateAccepted/In press - Jan 1 2019

Fingerprint

Pichia
Isoleucine
Labeling
Proteins
Costs and Cost Analysis
Isotopes
Biomolecular Nuclear Magnetic Resonance
Isotope Labeling
Costs
G-Protein-Coupled Receptors
Actins
Membrane Proteins
Yeast
Yeasts
Nuclear magnetic resonance spectroscopy
Molecular Weight
Labels
Bacteria
Molecular weight
Nuclear magnetic resonance

Keywords

  • C methyl labeling
  • Deuteration
  • Eukaryotic protein expression
  • Methyl TROSY
  • Pichia pastoris expression

ASJC Scopus subject areas

  • Biochemistry
  • Spectroscopy

Cite this

Improved strategy for isoleucine 1H/13C methyl labeling in Pichia pastoris. / Ali, Rustam; Clark, Lindsay D.; Zahm, Jacob A.; Lemoff, Andrew; Ramesh, Karthik; Rosenbaum, Daniel M.; Rosen, Michael K.

In: Journal of Biomolecular NMR, 01.01.2019.

Research output: Contribution to journalArticle

Ali, Rustam ; Clark, Lindsay D. ; Zahm, Jacob A. ; Lemoff, Andrew ; Ramesh, Karthik ; Rosenbaum, Daniel M. ; Rosen, Michael K. / Improved strategy for isoleucine 1H/13C methyl labeling in Pichia pastoris. In: Journal of Biomolecular NMR. 2019.
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