Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris

Lindsay Clark, Jacob A. Zahm, Rustam Ali, Maciej Kukula, Liangqiao Bian, Steven M. Patrie, Kevin H. Gardner, Michael Rosen, Daniel M. Rosenbaum*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

Abstract

13C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific 13C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient 13C isoleucine d1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets.

Original languageEnglish (US)
Pages (from-to)239-245
Number of pages7
JournalJournal of Biomolecular NMR
Volume62
Issue number3
DOIs
StatePublished - Jul 1 2015

Keywords

  • Actin
  • Deuteration
  • Eukaryotic expression system
  • Methyl labeling
  • Pichia pastoris
  • TROSY

ASJC Scopus subject areas

  • Biochemistry
  • Spectroscopy

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