title: Escherichia coli auxotroph host strains for amino acid-selective isotope labeling of recombinant proteins.
abstract: Enrichment of proteins with isotopes such as (2)H, (15)N, and (13)C 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.
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
authors.forename: Myat T
authors.initials: MT
authors.lastname: Lin
authors.affiliation: Department of Biochemistry and Molecular Biology, Nippon Medical School, Bunkyo-ku, Tokyo, Japan.
authors.forename: Risako
authors.initials: R
authors.lastname: Fukazawa
authors.affiliation: Department of Biochemistry and Molecular Biology, Nippon Medical School, Bunkyo-ku, Tokyo, Japan.
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Miyajima-Nakano
authors.affiliation: Department of Biochemistry and Molecular Biology, Nippon Medical School, Bunkyo-ku, Tokyo, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Matsushita
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
authors.forename: Sylvia K
authors.initials: SK
authors.lastname: Choi
authors.affiliation: Department of Biochemistry and Molecular Biology, Nippon Medical School, Bunkyo-ku, Tokyo, Japan. Electronic address: tiwasaki@nms.ac.jp.
authors.forename: Toshio
authors.initials: T
authors.lastname: Iwasaki
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007553:Isotope Labeling; D011994:Recombinant Proteins / Q000737:chemistry; D013045:Species Specificity
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins
keywords: Amino acids; Auxotrophs; Escherichia coli; Protein expression; Selective isotope labeling