title: Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system.
abstract: Using the single-protein-production (SPP) system, a protein of interest can be exclusively produced in high yield from its ACA-less gene in Escherichia coli expressing MazF, an ACA-specific mRNA interferase. It is thus feasible to study a membrane protein by solid-state NMR (SSNMR) directly in natural membrane fractions. In developing isotope-enrichment methods, we observed that (13)C was also incorporated into phospholipids, generating spurious signals in SSNMR spectra. Notable, with the SPP system a protein can be produced in total absence of cell growth caused by antibiotics. Here, we demonstrate that cerulenin, an inhibitor of phospholipid biosynthesis, can suppress isotope incorporation in the lipids without affecting membrane protein yield in the SPP system. SSNMR analysis of ATP synthase subunit c, an E. coli inner membrane protein, produced by the SPP method using cerulenin revealed that (13)C resonance signals from phospholipid were markedly reduced, while signals for the isotope-enriched protein were clearly present.
authors.affiliation: Department of Biochemistry, Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854, USA.
authors.forename: Lili
authors.initials: L
authors.lastname: Mao
authors.forename: Koichi
authors.initials: K
authors.lastname: Inoue
authors.forename: Yisong
authors.initials: Y
authors.lastname: Tao
authors.forename: Gaetano T
authors.initials: GT
authors.lastname: Montelione
authors.forename: Ann E
authors.initials: AE
authors.lastname: McDermott
authors.forename: Masayori
authors.initials: M
authors.lastname: Inouye
mesh_terms: D002569:Cerulenin / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010743:Phospholipids / Q000737:chemistry*
chemical_list: D008565:Membrane Proteins; D010743:Phospholipids; D002569:Cerulenin