RRC ID 52858
Author Lin MT, Sperling LJ, Frericks Schmidt HL, Tang M, Samoilova RI, Kumasaka T, Iwasaki T, Dikanov SA, Rienstra CM, Gennis RB.
Title A rapid and robust method for selective isotope labeling of proteins.
Journal Methods
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.
Volume 55(4)
Pages 370-8
Published 2011-12-1
DOI 10.1016/j.ymeth.2011.08.019
PII S1046-2023(11)00164-2
PMID 21925267
PMC PMC3261321
MeSH Amino Acid Motifs Binding Sites Electron Spin Resonance Spectroscopy Electron Transport Complex IV / biosynthesis* Electron Transport Complex IV / chemistry Escherichia coli / genetics Escherichia coli Proteins / biosynthesis* Escherichia coli Proteins / chemistry Ferredoxins / biosynthesis* Ferredoxins / chemistry Hydrogen Bonding Iron / chemistry Isotope Labeling / methods* Membrane Proteins / biosynthesis Membrane Proteins / chemistry Models, Molecular Nuclear Magnetic Resonance, Biomolecular Organisms, Genetically Modified Oxidation-Reduction Protein Subunits / biosynthesis Protein Subunits / chemistry Recombinant Proteins / biosynthesis Recombinant Proteins / chemistry Spectroscopy, Fourier Transform Infrared Sulfur / chemistry
IF 3.782
Times Cited 41
DNA material ML2 (RDB15927) ML3 (RDB15928) ML6 (RDB15929) ML8 (RDB15930) ML12 (RDB15931) ML14 (RDB15932) ML17 (RDB15933) ML21 (RDB15934) ML24 (RDB15935) ML25 (RDB15936) ML26 (RDB15937) ML31 (RDB15938) ML36 (RDB15939) ML40 (RDB15940) ML42 (RDB15941) ML43 (RDB15942).