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Efficient condensed-phase production of perdeuterated soluble and membrane proteins

  • William M. Schneider
  • Yuefeng Tang
  • S. Thangminlal Vaiphei
  • Lili Mao
  • Melissa Maglaqui
  • Masayori Inouye
  • Monica J. Roth
  • Gaetano T. Montelione
Article

Abstract

Protein perdeuteration approaches have tremendous value in protein NMR studies, but are limited by the high cost of perdeuterated media. Here, we demonstrate that E. coli cultures expressing proteins using either the condensed single protein production method (cSPP), or conventional pET expression plasmids, can be condensed prior to protein expression, thereby providing high-quality 2H, 13C, 15N-enriched protein samples at 2.5–10% the cost of traditional methods. As an example of the value of such inexpensively-produced perdeuterated proteins, we produced 2H, 13C, 15N-enriched E. coli cold shock protein A (CspA) and EnvZb in 40× condensed phase media, and obtained NMR spectra suitable for 3D structure determination. The cSPP system was also used to produce 2H, 13C, 15N-enriched E. coli plasma membrane protein YaiZ and outer membrane protein X (OmpX) in condensed phase. NMR spectra can be obtained for these membrane proteins produced in the cSPP system following simple detergent extraction, without extensive purification or reconstitution. This allows a membrane protein’s structural and functional properties to be characterized prior to reconstitution, or as a probe of the effects of subsequent purification steps on the structural integrity of membrane proteins. We also provide a standardized protocol for production of perdeuterated proteins using the cSPP system. The 10–40 fold reduction in costs of fermentation media provided by using a condensed culture system opens the door to many new applications for perdeuterated proteins in spectroscopic and crystallographic studies.

Keywords

Protein expression in E. coli Perdeuteration Protein NMR Single protein production system (SPP) Triple resonance NMR 

Notes

Acknowledgments

We thank Drs. Rajeswari Mani and Rong Xiao for helpful discussions and comments on the manuscript. This work was supported by the National Institutes of General Medical Science Protein Structure Initiative program, grants U54 GM074958 (to G.T.M and M.I.) and U54 GM75026 (G.T.M. and M.I.), and by grant RO1 GM070837 (to M.J.R.). W.M.S. was supported by NIH training grants T32 GM08360 and T32 A1007403.

Supplementary material

10969_2010_9083_MOESM1_ESM.pdf (420 kb)
Supplementary material 1 (PDF 420 kb)

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • William M. Schneider
    • 1
  • Yuefeng Tang
    • 2
  • S. Thangminlal Vaiphei
    • 1
    • 2
  • Lili Mao
    • 1
    • 2
  • Melissa Maglaqui
    • 2
  • Masayori Inouye
    • 1
    • 2
  • Monica J. Roth
    • 1
  • Gaetano T. Montelione
    • 1
    • 2
  1. 1.Department of Biochemistry, Robert Wood Johnson Medical SchoolUniversity of Medicine and Dentistry of New JerseyPiscatawayUSA
  2. 2.Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics ConsortiumRutgers UniversityPiscatawayUSA

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