Journal of Biomolecular NMR

, Volume 49, Issue 2, pp 131–137

Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system

  • Lili Mao
  • Koichi Inoue
  • Yisong Tao
  • Gaetano T. Montelione
  • Ann E. McDermott
  • Masayori Inouye
Article

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 13C 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 13C resonance signals from phospholipid were markedly reduced, while signals for the isotope-enriched protein were clearly present.

Keywords

cSPP Membrane protein Phospholipid biosynthesis Cerulenin SSNMR 

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Lili Mao
    • 1
    • 4
  • Koichi Inoue
    • 1
    • 4
  • Yisong Tao
    • 2
    • 4
  • Gaetano T. Montelione
    • 1
    • 3
    • 4
  • Ann E. McDermott
    • 2
    • 4
  • Masayori Inouye
    • 1
    • 4
  1. 1.Department of Biochemistry, Center for Advanced Biotechnology and MedicineRobert Wood Johnson Medical SchoolPiscatawayUSA
  2. 2.Department of ChemistryColumbia UniversityNew YorkUSA
  3. 3.Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and MedicineRutgers, The State University of New JerseyPiscatawayUSA
  4. 4.New York Center for Membrane Protein Structure (NYCOMPS)Rutgers UniversityPiscatawayUSA

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