Journal of Structural and Functional Genomics

, Volume 8, Issue 4, pp 167–172 | Cite as

Application of Mistic to improving the expression and membrane integration of histidine kinase receptors from Escherichia coli

  • Georgia Kefala
  • Witek Kwiatkowski
  • Luis Esquivies
  • Innokentiy Maslennikov
  • Senyon Choe


Integral membrane proteins have become the focus of interest of many laboratories and structural genomics consortia, but their study is hampered by bottlenecks in production, solubilization, purification and crystallization. In our laboratory we have addressed the problem of high-level protein expression in the membrane of Escherichia coli by use of Mistic, a novel Bacillus subtilis protein, as a fusion partner. In this study we examine the effect of Mistic on protein expression and membrane integration levels of members of the E. coli histidine kinase receptor family. We find that Mistic fusion invariably increases the overall yield by targeting the cargo proteins more efficiently to the membrane and may even replace the signal sequence. Mistic fusion methods will likely be instrumental for high-level expression of other integral membrane proteins.


Membrane protein Escherichia coli Protein expression Mistic Histidine kinase receptor 



Integral membrane protein


Signal peptide


Histidine kinase receptor


Polymerase chain reaction


Phosphate buffer saline


Mistic fused protein, misticated


Non-mistic fused, non-misticated


Full length




Multi-wavelength anomalous diffraction



We thank Tony Hunter and Jill Meisenhelder (Salk Institute) for providing facilities for, and help with the phosphorylation assays, and Kit Pogliano (UCSD) for providing the E. coli K-12 MG1655 strain. This work is supported by NIH Protein Structure Initiative grant GM074929 and GM74821.


  1. 1.
    Frishman D, Mewes HW (1997) Nat Struct Biol 4:626–628PubMedCrossRefGoogle Scholar
  2. 2.
    Kihara D, Kanehisa M (2000) Genome Res 10:731–743PubMedCrossRefGoogle Scholar
  3. 3.
    Raman P, Cherezov V, Caffrey M (2006) Cell Mol Life Sci 63:36–51PubMedCrossRefGoogle Scholar
  4. 4.
    Roosild TP, Greenwald J, Vega M, Castronovo S, Riek R, Choe S (2005) Science 307:1317–1321PubMedCrossRefGoogle Scholar
  5. 5.
    Wolanin PM, Thomason PA, Stock JB (2002) Genome Biol 3, REVIEWS3013Google Scholar
  6. 6.
    Loomis WF, Shaulsky G, Wang N (1997) J Cell Sci 110:1141–1145PubMedGoogle Scholar
  7. 7.
    Stock AM, Robinson VL, Goudreau PN (2000) Annu Rev Biochem 69:183–215PubMedCrossRefGoogle Scholar
  8. 8.
    Daley DO, Rapp M, Granseth E, Melen K, Drew D, von Heijne G (2005) Science 308:1321–1323PubMedCrossRefGoogle Scholar
  9. 9.
    Misra RV, Horler RS, Reindl W, Goryanin II, Thomas GH (2005) Nucleic Acids Res 33:D329–D333PubMedCrossRefGoogle Scholar
  10. 10.
    Médigue C, Viari A, Hénaut A, Danchin A (1993) Microbiol Rev 57:623–654PubMedGoogle Scholar
  11. 11.
    Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) J Mol Biol 340:783–795PubMedCrossRefGoogle Scholar
  12. 12.
    Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A (2003) Nucleic Acids Res 31:3784–3788PubMedCrossRefGoogle Scholar
  13. 13.
    Valent QA, de Gier JW, von Heijne G, Kendall DA, ten Hagen-Jongman CM, Oudega B, Luirink J (1997) Mol Microbiol 25:53–64PubMedCrossRefGoogle Scholar
  14. 14.
    Roosild TP, Vega M, Castronovo S, Choe S (2006) BMC Struct Biol 6:10PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Georgia Kefala
    • 1
  • Witek Kwiatkowski
    • 1
  • Luis Esquivies
    • 1
  • Innokentiy Maslennikov
    • 1
  • Senyon Choe
    • 1
  1. 1.Structural Biology LaboratoryThe Salk Institute for Biological StudiesLa JollaUSA

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