@article{d5ac2b1915f74cc4abf2875806836417,
title = "A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins",
abstract = "A selective protonation strategy is described that uses [3-2H] 13C α-ketoisovalerate to introduce (1H-δ methyl)-leucine and (1H-γ methyl)- valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of ≃100 mg/L α-ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] α-ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-δ1 methyl)- isoleucine (>90% incorporation), 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.",
keywords = "Deuteration, Methyl protonation, α-ketobutyrate, α-ketoisovalerate",
author = "Goto, {Natalie K.} and Gardner, {Kevin H.} and Mueller, {Geoffrey A.} and Willis, {Randall C.} and Kay, {Lewis E.}",
note = "Funding Information: This research was supported by a grant from the Medical Research Council of Canada. We would like to thank Grace DeSantis (Univiversity of Toronto) for her guidance in the amino acid derivatization reactions and Dr. Mike Rosen (Sloan-Kettering, New York) for his helpful advice. We also gratefully acknowledge the technical assistance provided by Dr. Alex Young (Univiversity of Toronto) with the GC-MS analyses and Rey Interior (Hospital for Sick Children Biotechnology Center) for performing the protein hydrolysis. The authors are grateful to Cambridge Isotope Laboratories (Andover, MA) for supplying α-ketoisovalerate free of charge. K.H.G. gratefully acknowledges a postdoctoral fellowship from the Helen Hay Whitney Foundation and N.K.G. is a recipient of an NSERC postgraduate scholarship. L.E.K. is an International Howard Hughes Research Scholar.",
year = "1999",
doi = "10.1023/A:1008393201236",
language = "English (US)",
volume = "13",
pages = "369--374",
journal = "Journal of Biomolecular NMR",
issn = "0925-2738",
publisher = "Springer Netherlands",
number = "4",
}