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Systems for the Cell-Free Synthesis of Proteins

  • Lei Kai
  • Christian Roos
  • Stefan Haberstock
  • Davide Proverbio
  • Yi Ma
  • Friederike Junge
  • Mikhail Karbyshev
  • Volker Dötsch
  • Frank BernhardEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 800)

Abstract

We describe a system for the cell-free expression of proteins based on extracts from Escherichia coli. Two reaction configurations, batch and continuous exchange, are discussed and analytical scale as well as preparative scale setups are documented. Guidelines for the systematic development and optimization of cell-free expression protocols are given in detail. We further provide specific protocols and parameters for the cell-free production of membrane proteins. High-throughput screening applications of CF expression systems are exemplified as new tools for genomics and proteomics studies.

Key words

Cell-free expression Membrane proteins Expression optimization Detergents Liposomes Cell extracts 

References

  1. 1.
    Cell-free protein production, methods and protocols (2010) Y. Endo, K. Takai, T. Ueda, (eds.). Methods Mol. Biol. 607, Humana Press, New York.Google Scholar
  2. 2.
    Schwarz D, Junge F, Durst F, et al (2007) Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems. Nat Protoc 2: 2945–2957PubMedCrossRefGoogle Scholar
  3. 3.
    Jung, F, Schneider B, Reckel S, Schwarz D, Dötsch V, Bernhard F (2008) Large-scale production of functional membrane proteins. Cell Mol. Life Sci 65: 1729–1755CrossRefGoogle Scholar
  4. 4.
    Katzen F, Kudlicki W (2006) Efficient generation of insect-based cell-free translation extracts active in glycosylation and signal sequence processing. J Biotechnol 125: 194–197PubMedCrossRefGoogle Scholar
  5. 5.
    Zubay G (1973) In vitro synthesis of protein in microbial systems. Annu Rev Genet 7: 267–287PubMedCrossRefGoogle Scholar
  6. 6.
    Mureev S, Kovtun O, Nguyen UT, Alexandrov K (2009) Species-independent translational leaders facilitate cell-free expression. Nat Biotechnol 27: 747–752PubMedCrossRefGoogle Scholar
  7. 7.
    Vinarov DA, Loushin Newman CL, Markley J L (2006) Wheat germ cell-free platform for eukaryotic protein production. FEBS J 273: 4160–4169PubMedCrossRefGoogle Scholar
  8. 8.
    Swartz J (2006) Developing cell-free biology for industrial applications. J Ind Microbiol Biotechnol 33: 476–485PubMedCrossRefGoogle Scholar
  9. 9.
    Kim TW, Kim DM, Choi CY (2006) Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system. J Biotechnol 124: 373–380PubMedCrossRefGoogle Scholar
  10. 10.
    Spirin AS, Baranov, VI, Ryabova LA, Ovodov SY, Alakhov YB (1988) A continuous cell-free translation system capable of producing polypeptides in high yield. Science 242: 1162–1164PubMedCrossRefGoogle Scholar
  11. 11.
    Kigawa T, Yokoyama S (1991) A continuous cell-free protein synthesis system for coupled transcription-translation. J Biochem 110: 166–168PubMedGoogle Scholar
  12. 12.
    Sitaraman K, Chatterjee DK (2009) High-throughput protein expression using cell-free system. Methods Mol. Biol 498: 229–244PubMedCrossRefGoogle Scholar
  13. 13.
    Aoki M, Matsuda T, Tomo Y, Miyata Y, Inoue M, Kigawa T, Yokoyama S (2009) Automated system for high-throughput protein production using the dialysis cell-free method. Protein Expr. Purif 68: 128–136PubMedCrossRefGoogle Scholar
  14. 14.
    Junge F, Luh LM, Proverbio D, et al (2010) Modulation of G-protein coupled receptor sample quality by modified cell-free expression protocols: a case study of the human endothelin A receptor. J Struct Biol 172: 94–106PubMedCrossRefGoogle Scholar
  15. 15.
    Kai L, Kaldenhoff R, Lian J, Zhu X, Dötsch V, Bernhard F, Cen P, Xu Z (2010) Preparative scale production of functional mouse aquaporin 4 using different cell-free expression modes. PLoS ONE doi: 10.1371/journal.pone.0012972
  16. 16.
    Hovijitra NT, Wuu JJ, Peaker B, Swartz JR (2009) Cell-free synthesis of functional aquaporin Z in synthetic liposomes. Biotechnol Bioeng 104: 40–49PubMedCrossRefGoogle Scholar
  17. 17.
    Berrier C, Guilvout I, Bayan N, Park KH, Mesneau A, Chami M, Pugsley AP, Ghazi A (2010) Coupled cell-free synthesis and lipid vesicle insertion of a functional oligomeric channel MscL. MscL does not need the insertase YidC for insertion in vitro. Biochim Biophys Acta 1808: 41–46PubMedGoogle Scholar
  18. 18.
    Schwarz D, Daley D, Beckhaus T, Dötsch V, Bernhard F (2010) Cell-free expression profiling of E. coli inner membrane proteins. Proteomics 10:P 1762–1779Google Scholar
  19. 19.
    Reckel S, Sobhanifar S, Schneider B, et al (2008) Transmembrane segment enhanced labeling as a tool for the backbone assignment of -helical membrane proteins. PNAS 105: 8262–8267PubMedCrossRefGoogle Scholar
  20. 20.
    Apponyi MA, Ozawa K, Dixon NE, Otting G (2008) Cell-free protein synthesis for analysis by NMR spectroscopy. Methods Mol Biol 426: 257–268PubMedCrossRefGoogle Scholar
  21. 21.
    Li Y, Wang E, Wang Y (1999) A modified procedure for fast purification of T7 RNA polymerase. Protein Expr Purif 16: 355–358PubMedCrossRefGoogle Scholar
  22. 22.
    Drew DE, von Heijne G, Nordlund P, de Gier JW (2001) Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli. FEBS Lett 507: 220–224PubMedCrossRefGoogle Scholar
  23. 23.
    Yabuki T, Motoda Y, Hanada K, et al (2007) A robust two-step PCR method of template DNA production for high-throughput cell-free protein synthesis. J Struct Funct Genomics 8: 173–191PubMedCrossRefGoogle Scholar
  24. 24.
    Schneider B, Junge F, Shirokov VA, Durst F, Schwarz D, Dötsch V, Bernhard F (2010) Membrane protein expression in cell-free systems. Methods Mol Biol 601: 165–186PubMedCrossRefGoogle Scholar
  25. 25.
    Shimono K, Goto M, Kikukawa T, Miyauchi S, Shirouzu M, Kamo N, Yokoyama S (2009) Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid. Protein Sci 18: 2160–2171PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Lei Kai
    • 1
  • Christian Roos
    • 1
  • Stefan Haberstock
    • 1
  • Davide Proverbio
    • 1
  • Yi Ma
    • 2
  • Friederike Junge
    • 1
  • Mikhail Karbyshev
    • 1
  • Volker Dötsch
    • 1
  • Frank Bernhard
    • 1
    Email author
  1. 1.Centre for Biomolecular Magnetic Resonance, Institute for Biophysical ChemistryGoethe-University of Frankfurt/MainFrankfurt/MainGermany
  2. 2.School of Bioscience and BioengineeringSouth China University of TechnologyGuangzhouChina

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