title: Selective labeling of a membrane peptide with 15N-amino acids using cells grown in rich medium.
abstract: Nuclear magnetic resonance spectra of membrane proteins containing multiple transmembrane helices have proven difficult to resolve due to the redundancy of aliphatic and Ser/Thr residues in transmembrane domains and the low chemical shift dispersity exhibited by residues in alpha-helical structures. Although (13)C- and (15)N-labeling are useful tools in the biophysical analysis of proteins, selective labeling of individual amino acids has been used to help elucidate more complete structures and to probe ligand-protein interactions. In general, selective labeling has been performed in Escherichia coli expression systems using minimal media supplemented with a single labeled amino acid and nineteen other unlabeled amino acids and/or by using auxotrophs for specific amino acids. Growth in minimal media often results in low yields of cells or expression products. We demonstrate a method in which one labeled amino acid is added to a rich medium. These conditions resulted in high expression (> or =100 mg/L) of a test fusion protein and milligram quantities of the selectively labeled membrane peptide after cyanogen bromide cleavage to release the peptide from the fusion protein. High levels of (15)N incorporation and acceptable levels of cross-labeling into other amino acid residues of the peptide were achieved. Growth in rich media is a simple and convenient alternative to growth in supplemented minimal media and is readily applicable to the expression of proteins selectively labeled with specific amino acids.
authors.affiliation: Department of Chemistry and the Macromolecular Assemblies Institute, The College of Staten Island of the City University of New York, Staten Island, NY 10314, USA.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Englander
authors.forename: Leah
authors.initials: L
authors.lastname: Cohen
authors.forename: Boris
authors.initials: B
authors.lastname: Arshava
authors.forename: Racha
authors.initials: R
authors.lastname: Estephan
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Becker
authors.forename: Fred
authors.initials: F
authors.lastname: Naider
mesh_terms: D000596:Amino Acids / Q000032:analysis* / Q000378:metabolism; D003470:Culture Media; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry
chemical_list: D000596:Amino Acids; D003470:Culture Media; D008565:Membrane Proteins; D009587:Nitrogen Isotopes; D010455:Peptides; D011993:Recombinant Fusion Proteins