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 (13)C- and (15)N-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.
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States.
authors.forename: Myat T
authors.initials: MT
authors.lastname: Lin
authors.forename: Lindsay J
authors.initials: LJ
authors.lastname: Sperling
authors.forename: Heather L
authors.initials: HL
authors.lastname: Frericks Schmidt
authors.forename: Ming
authors.initials: M
authors.lastname: Tang
authors.forename: Rimma I
authors.initials: RI
authors.lastname: Samoilova
authors.forename: Takashi
authors.initials: T
authors.lastname: Kumasaka
authors.forename: Toshio
authors.initials: T
authors.lastname: Iwasaki
authors.forename: Sergei A
authors.initials: SA
authors.lastname: Dikanov
authors.forename: Chad M
authors.initials: CM
authors.lastname: Rienstra
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000096:biosynthesis* / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry; D005288:Ferredoxins / Q000096:biosynthesis* / Q000737:chemistry; D006860:Hydrogen Bonding; D007501:Iron / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D030781:Organisms, Genetically Modified; D010084:Oxidation-Reduction; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013455:Sulfur / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D005288:Ferredoxins; D008565:Membrane Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D013455:Sulfur; D007501:Iron; C510605:CyoA protein, E coli; D003576:Electron Transport Complex IV