Escherichia coli Auxotroph Host Strains for Amino Acid-Selective Isotope Labeling of Recombinant Proteins

Myat T. Lin, Risako Fukazawa, Yoshiharu Miyajima-Nakano, Shinichi Matsushita, Sylvia K. Choi, Toshio Iwasaki, Robert B. Gennis

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Enrichment of proteins with isotopes such as 2H, 15N, and 13C is commonly carried out in magnetic resonance and vibrational spectroscopic characterization of protein structures, mechanisms, and dynamics. Although uniform isotopic labeling of proteins is straightforward, efficient labeling of proteins with only a selected set of amino acid types is often challenging. A number of approaches have been described in the literature for amino acid-selective isotope labeling of proteins, each with its own limitations. Since Escherichia coli represents the most cost-effective and widely used host for heterologous production of foreign proteins, an efficient method to express proteins selectively labeled with isotopes would be highly valuable for these studies. However, an obvious drawback is misincorporation and dilution of input isotope labels to unwanted amino acid types due to metabolic scrambling in vivo. To overcome this problem, we have generated E. coli auxotroph strains that are compatible with the widely used T7 RNA polymerase overexpression systems and that minimize metabolic scrambling. We present several examples of selective amino acid isotope labeling of simple and complex proteins with bound cofactors, as an initial guide for practical applications of these E. coli strains.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages45-66
Number of pages22
DOIs
StatePublished - Jan 1 2015

Publication series

NameMethods in Enzymology
Volume565
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Lin, M. T., Fukazawa, R., Miyajima-Nakano, Y., Matsushita, S., Choi, S. K., Iwasaki, T., & Gennis, R. B. (2015). Escherichia coli Auxotroph Host Strains for Amino Acid-Selective Isotope Labeling of Recombinant Proteins. In Methods in Enzymology (pp. 45-66). (Methods in Enzymology; Vol. 565). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2015.05.012