Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris
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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 δ1-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.
KeywordsMethyl labeling TROSY Eukaryotic expression system Pichia pastoris Deuteration Actin
Funding was provided by a National Science Foundation Predoctoral Fellowship (Grant No. 1000136529 to L.C.), the Welch Foundation (I-1770 to D.M.R, I-1544 to M.K.R., I-1424 to K.H.G.), the Searle Scholars Program (D.M.R), a Packard Foundation Fellowship (D.M.R), the National Institutes of Health (T32 GM008297 supporting J.Z., R01 GM106239 to K.H.G., R01-GM56322 to M.K.R) and the Howard Hughes Medical Institute (M.K.R.).