@article{ef9fd7c71a9940e9ac7579c84a622fd9,
title = "A rapid and robust method for selective isotope labeling of proteins",
abstract = "Amino-acid selective isotope labeling of proteins offers numerous advantages in mechanistic studies by revealing structural and functional information unattainable from a crystallographic approach. However, efficient labeling of proteins with selected amino acids necessitates auxotrophic hosts, which are often not available. We have constructed a set of auxotrophs in a commonly used Escherichia coli expression strain C43(DE3), a derivative of E. coli BL21(DE3), which can be used for isotopic labeling of individual amino acids or sets of amino acids. These strains have general applicability to either soluble or membrane proteins that can be expressed in E. coli. We present examples in which proteins are selectively labeled with 13C- and 15N-amino acids and studied using magic-angle spinning solid-state NMR and pulsed EPR, demonstrating the utility of these strains for biophysical characterization of membrane proteins, radical-generating enzymes and metalloproteins.",
keywords = "Auxotroph, EPR, Escherichia coli, Isotope, NMR, Protein expression",
author = "Lin, {Myat T.} and Sperling, {Lindsay J.} and {Frericks Schmidt}, {Heather L.} and Ming Tang and Samoilova, {Rimma I.} and Takashi Kumasaka and Toshio Iwasaki and Dikanov, {Sergei A.} and Chad Rienstra and Gennis, {Robert B.}",
note = "Funding Information: We thank Dr. Jihyun F. Kim from the Systems Microbiology Research Center, KRIBB, Daejeon 305-806, South Korea for providing the genome sequence of E. coli BL21(DE3). We thank Dr. John E. Cronan and Dr. Nicholas R. De Lay from the Department of Microbiology at the University of Illinois at Urbana-Champaign for providing the pKD46, pKD3, pKD4 and pCP20 plasmids. We thank Dr. Deborah Berthold from the Department of Chemistry at the University of Illinois at Urbana-Champaign for advice on the manuscript. This work was supported by the DE-FG02-87ER13716 (R.B.G) and DE-FG02-08ER15960 (S.A.D) Grants from US Department of Energy , National Institute of Health & NIGMS Roadmap Initiative ( R01GM075937 ), the JSPS Grant-in-aid 21659111 (T.I), the International Collaborations in Chemistry Grant from JSPS (T.I) and NSF (CHE-1026541 to S.A.D), NIH grant GM062954 (S.A.D), and the NIH Molecular Biophysics Training Grant ( PHS 5 T32 GM008276 ) and the Ullyot Fellowship to L.J.S. ",
year = "2011",
month = dec,
doi = "10.1016/j.ymeth.2011.08.019",
language = "English (US)",
volume = "55",
pages = "370--378",
journal = "Methods",
issn = "1046-2023",
publisher = "Academic Press Inc.",
number = "4",
}