Journal of Structural and Functional Genomics

, Volume 10, Issue 3, pp 219–225 | Cite as

Independently inducible system of gene expression for condensed single protein production (cSPP) suitable for high efficiency isotope enrichment

  • William M. Schneider
  • Masayori Inouye
  • Gaetano T. Montelione
  • Monica J. Roth
Article

Abstract

The ability to produce isotope-enriched proteins is fundamental to the success of modern protein NMR, and is particularly essential for NMR activities in structural genomics projects. Conventional methods of protein production often prove to be cost prohibitive for obtaining samples, particularly perdeuterated and site-specifically labeled proteins. The condensed single protein production system (cSPP), providing protein expression following condensation of cells 10–40 fold, allows for the production of such samples at a fraction of the cost. The previously described cSPP system is a two plasmid system where both the MazF toxin and ACA-less target gene are coinduced with IPTG. Coinduction results in 10–20% of the target protein produced without isotopic enrichment. Though the unlabeled protein is generally not visible in isotope-filtered NMR experiments, it results in an effective reduction in yield of the observable sample. By altering the cSPP system and separating the induction of the MazF toxin, required to convert cells into a semiquiescent state prior to condensation, from the expression of the target gene, we are now able to eliminate the unlabeled protein fraction and improve the isotope incorporation. Here we describe a series of pCold(tet) vectors with various features that can be used in the dual inducible cSPP(tet) system to obtain high-quality isotopically enriched protein at as little as 2.5% the cost of traditional methods.

Keywords

Anhydrotetracycline Isotope labeling MuLV IN pCold vectors Structural genomics Triple resonance NMR 

Abbreviations

cSPP

Condensed single protein production

M-MuLV IN

Moloney Murine Leukemia Virus Integrase

TEE

Translation enhancing element

Notes

Acknowledgements

We thank M. Suzuki, L. Mao, Y. Tang, and P. Rossi for helpful discussions in the course of this work, and for their comments on the manuscript. This work was supported by the National Institutes Health Grants RO1 GM070837 (to M.J.R.), U54 GM074958 (G.T.M. and M.I.), U54 GM75026 (G.T.M. and M.I.), 1R01 GM085449 (M.I.). W.M.S. was supported by NIH training grants T32 GM08360 and T32 A1007403.

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Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • William M. Schneider
    • 1
  • Masayori Inouye
    • 2
    • 3
    • 4
  • Gaetano T. Montelione
    • 2
    • 3
    • 4
    • 5
  • Monica J. Roth
    • 1
  1. 1.Department of BiochemistryUniversity of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical SchoolPiscatawayUSA
  2. 2.Department of BiochemistryUniversity of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical SchoolPiscatawayUSA
  3. 3.New York Consortium for Membrane Protein Structure (NYCOMPS)PiscatawayUSA
  4. 4.Northeast Structural Genomics ConsortiumPiscatawayUSA
  5. 5.Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and MedicineRutgers UniversityPiscatawayUSA

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