title: Selective methyl labeling of eukaryotic membrane proteins using cell-free expression.
abstract: Structural characterization of membrane proteins and other large proteins with NMR relies increasingly on perdeuteration combined with incorporation of specifically protonated amino acid moieties, such as methyl groups of isoleucines, valines, or leucines. The resulting proton dilution reduces dipolar broadening producing sharper resonance lines, ameliorates spectral crowding, and enables measuring of crucial distances between and to methyl groups. While incorporation of specific methyl labeling is now well established for bacterial expression using suitable precursors, corresponding methods are still lacking for cell-free expression, which is often the only choice for producing labeled eukaryotic membrane proteins in mg quantities. Here we show that we can express methyl-labeled human integral membrane proteins cost-effectively by cell-free expression based of crude hydrolyzed ILV-labeled OmpX inclusion bodies. These are obtained in Escherichia coli with very high quantity and represent an optimal intermediate to channel ILV precursors into the eukaryotic proteins.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School , Boston, Massachusetts 02115, United States.
authors.forename: Rasmus
authors.initials: R
authors.lastname: Linser
authors.forename: Vladimir
authors.initials: V
authors.lastname: Gelev
authors.forename: Franz
authors.initials: F
authors.lastname: Hagn
authors.forename: Haribabu
authors.initials: H
authors.lastname: Arthanari
authors.forename: Sven G
authors.initials: SG
authors.lastname: Hyberts
authors.forename: Gerhard
authors.initials: G
authors.lastname: Wagner
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry; D002474:Cell-Free System; D003362:Cost-Benefit Analysis; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D006867:Hydrolases / Q000378:metabolism; D006868:Hydrolysis; D002479:Inclusion Bodies / Q000378:metabolism; D007532:Isoleucine / Q000737:chemistry; D007930:Leucine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D014633:Valine / Q000737:chemistry
chemical_list: D001425:Bacterial Outer Membrane Proteins; D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D008823:Micelles; D007532:Isoleucine; C482905:OmpX protein, E coli; D006859:Hydrogen; D006867:Hydrolases; D007930:Leucine; D014633:Valine