title: Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements.
abstract: Nuclear magnetic resonance (NMR) is a powerful technique for solving protein structures or studying their interactions. However, it requires molecules labeled with NMR sensitive isotopes like carbon (13)C and nitrogen (15)N. The recombinant expression of labeled proteins is simple to perform but requires quite expensive chemicals. When there is a need for special labeled chemicals, like uniformly (13)C-labeled myristic acid, the price significantly rises. Here we describe a cost-effective method for the recombinant expression of uniformly labeled myristoylated proteins in Escherichia coli demonstrated on the production of Mason-Pfizer monkey virus matrix protein. We used the ability of E. coli to naturally synthetize myristic acid. When grown in isotopically labeled medium the myristic acid will be labelled as well. Bacteria were co-transfected with plasmid carrying gene for yeast N-myristoyltransferase which ensures myristoylation of expressed protein. This process provided 1.8mg of the myristoylated, doubly labeled ((13)C/(15)N)M-PMV matrix protein from 1L of (15)N/(13)C labeled M9 medium. The price represents approximately 50% cost reduction of conventional method using commercially available [U-(13)C]myristic acid.
authors.affiliation: Laboratory of NMR Spectroscopy, Institute of Chemical Technology, Prague, Technicka 5, 166 28 Prague 6, Czech Republic; Department of Biochemistry and Microbiology, Institute of Chemical Technology,Prague, Technicka 5, 166 28 Prague 6, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Kroupa
authors.affiliation: Laboratory of NMR Spectroscopy, Institute of Chemical Technology, Prague, Technicka 5, 166 28 Prague 6, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Prchal
authors.affiliation: Laboratory of NMR Spectroscopy, Institute of Chemical Technology, Prague, Technicka 5, 166 28 Prague 6, Czech Republic; Department of Biochemistry and Microbiology, Institute of Chemical Technology,Prague, Technicka 5, 166 28 Prague 6, Czech Republic.
authors.forename: Michal
authors.initials: M
authors.lastname: Doležal
authors.affiliation: Department of Biochemistry and Microbiology, Institute of Chemical Technology,Prague, Technicka 5, 166 28 Prague 6, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Ruml
authors.affiliation: Laboratory of NMR Spectroscopy, Institute of Chemical Technology, Prague, Technicka 5, 166 28 Prague 6, Czech Republic. Electronic address: hrabalr@vscht.cz.
authors.forename: Richard
authors.initials: R
authors.lastname: Hrabal
mesh_terms: D000215:Acylation; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007553:Isotope Labeling / Q000191:economics / Q000379:methods; D016093:Mason-Pfizer monkey virus / Q000235:genetics; D019814:Myristic Acid / Q000737:chemistry / Q000378:metabolism; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014162:Transfection; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins; D019814:Myristic Acid; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase
keywords: Isotopic labeling; M-PMV; Matrix protein; N-terminal myristoylation; NMR