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

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 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.

• Content Type Journal Article
• Pages 1-7
• DOI 10.1007/s10858-011-9469-5
• Authors
  • Lili Mao, Department of Biochemistry, Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854, USA
  • Koichi Inoue, Department of Biochemistry, Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854, USA
  • Yisong Tao, Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA
  • Gaetano T. Montelione, Department of Biochemistry, Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854, USA
  • Ann E. McDermott, Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA
  • Masayori Inouye, Department of Biochemistry, Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854, USA

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738

Source: Journal of Biomolecular NMR