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


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.


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



Condensed single protein production


Moloney Murine Leukemia Virus Integrase


Translation enhancing element



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.


  1. 1.
    Suzuki M, Mao L, Inouye M (2007) Single protein production (SPP) system in Escherichia coli. Nat Protoc 2:1802–1810PubMedCrossRefGoogle Scholar
  2. 2.
    Suzuki M, Rohini R, Zheng H, Woychik N, Inouye M (2006) Bacterial bioreactors for high yield production of recombinant protein. J Biol Chem 281:37559–37565PubMedCrossRefGoogle Scholar
  3. 3.
    Suzuki M, Zhang J, Liu M, Woychik NA, Inouye M (2005) Single protein production in living cells facilitated by an mRNA interferase. Mol Cell 18:253–261PubMedCrossRefGoogle Scholar
  4. 4.
    Qing G, Ma L-C, Khorchid A, Swapna GVT, Mal TK, Takayama MM, Xia B, Phadtare S, Ke H, Acton T et al (2004) Cold-shock induced high-yield protein production in Escherichia coli. Nat Biotech 22:877–882CrossRefGoogle Scholar
  5. 5.
    Maloy SR, Stewart VJ, Taylor RK (1996) Genetic analysis of pathogenic bacteria: a laboratory manual. Cold Spring Harbor Laboratory Press, NYGoogle Scholar
  6. 6.
    Holler TP, Foltin SK, Ye QZ, Hupe DJ (1993) HIV1 integrase expressed in Escherichia coli from a synthetic gene. Gene 136:323–328PubMedCrossRefGoogle Scholar
  7. 7.
    Etchegaray J-P, Inouye M (1999) Translational enhancement by and element downstream of the initiation codon in Escherichia coli. J Biol Chem 274:10079–10085PubMedCrossRefGoogle Scholar
  8. 8.
    Clore GM, Omichinski JG, Sakaguchi K, Zambrano N, Sakamoto H, Appella E, Gronenborn AM (1994) High-resolution structure of the oligomerization domain of p53 by multidimensional NMR. Science 265:386–391PubMedCrossRefGoogle Scholar
  9. 9.
    Ikura M, Bax A (1992) Isotope-filtered 2D NMR of a protein peptide complex—study of a skeletal-muscle myosin light chain kinase fragment bound to calmodulin. J Am Chem Soc 114:2433–2440CrossRefGoogle Scholar

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

Personalised recommendations