Characterization and Production of Protein Complexes by Co-expression in Escherichia coli

  • Matthias Haffke
  • Martin Marek
  • Martin Pelosse
  • Marie-Laure Diebold
  • Uwe Schlattner
  • Imre Berger
  • Christophe Romier
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1261)

Abstract

The functional units within cells are often macromolecular complexes rather than single species. Production of these complexes as assembled homogenous samples is a prerequisite for their biophysical and structural characterization and hence an understanding of their function in molecular terms. Co-expression in Escherichia coli has been used routinely to decipher the subunit composition, assembly, and production of whole protein complexes. Such complexes can then be used to reconstitute protein/nucleic acid complexes in vitro. In this chapter we present protocols for the widely utilized ACEMBL and pET-MCN/pET-MCP vector series which enable the rapid and automated co-expression of protein complexes in Escherichia coli.

Key words

Protein complexes Co-expression Escherichia coli ACEMBL pET-MCN pET-MCP Cloning SLIC Plasmid concatenation Expression tests High throughput Automation 

References

  1. 1.
    Kerrigan JJ, Xie Q, Ames RS, Lu Q (2011) Production of protein complexes via co-expression. Protein Expr Purif 75:1–14PubMedCrossRefGoogle Scholar
  2. 2.
    Vincentelli R, Romier C (2013) Expression in Escherichia coli: becoming faster and more complex. Curr Opin Struct Biol 23:326–334PubMedCrossRefGoogle Scholar
  3. 3.
    Perrakis A, Romier C (2008) Assembly of protein complexes by coexpression in prokaryotic and eukaryotic hosts: an overview. Methods Mol Biol 426:247–256PubMedCrossRefGoogle Scholar
  4. 4.
    Romier C (2008) Protein complexes assembly by multi-expression in bacterial and eukaryotic hosts. In: Sussman J (ed) Structural proteomics. World Scientific Publishing Company, London, pp 233–250Google Scholar
  5. 5.
    Romier C, Ben JM, Albeck S et al (2006) Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies. Acta Crystallogr D Biol Crystallogr 62:1232–1242PubMedCrossRefGoogle Scholar
  6. 6.
    An Y, Meresse P, Mas PJ, Hart DJ (2011) CoESPRIT: a library-based construct screening method for identification and expression of soluble protein complexes. PLoS One 6:e16261PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Bieniossek C, Nie Y, Frey D et al (2009) Automated unrestricted multigene recombineering for multiprotein complex production. Nat Methods 6:447–450PubMedCrossRefGoogle Scholar
  8. 8.
    Diebold ML, Fribourg S, Koch M et al (2011) Deciphering correct strategies for multiprotein complex assembly by co-expression: application to complexes as large as the histone octamer. J Struct Biol 175:178–188PubMedCrossRefGoogle Scholar
  9. 9.
    Fribourg S, Romier C, Werten S et al (2001) Dissecting the interaction network of multiprotein complexes by pairwise coexpression of subunits in E. coli. J Mol Biol 306:363–373PubMedCrossRefGoogle Scholar
  10. 10.
    Held D, Yaeger K, Novy R (2003) New coexpression vectors for expanded compatibilities in E. coli. inNovations 18:4–6Google Scholar
  11. 11.
    Novy R, Yaeger K, Held D, Mierendorf R (2002) Coexpression of multiple target proteins in E. coli. inNovations 15:2–6Google Scholar
  12. 12.
    Scheich C, Kummel D, Soumailakakis D et al (2007) Vectors for co-expression of an unrestricted number of proteins. Nucleic Acids Res 35:e43PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Selleck W, Tan S (2008) Recombinant protein complex expression in E. coli. Curr Protoc Protein Sci. Chapter 5, Unit 5 21Google Scholar
  14. 14.
    Tan S (2001) A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli. Protein Expr Purif 21:224–234PubMedCrossRefGoogle Scholar
  15. 15.
    Tan S, Kern RC, Selleck W (2005) The pST44 polycistronic expression system for producing protein complexes in Escherichia coli. Protein Expr Purif 40:385–395PubMedCrossRefGoogle Scholar
  16. 16.
    Tolia NH, Joshua-Tor L (2006) Strategies for protein coexpression in Escherichia coli. Nat Methods 3:55–64PubMedCrossRefGoogle Scholar
  17. 17.
    Lariviere L, Plaschka C, Seizl M et al (2012) Structure of the Mediator head module. Nature 492:448–451PubMedCrossRefGoogle Scholar
  18. 18.
    Busso D, Peleg Y, Heidebrecht T et al (2011) Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study. J Struct Biol 175:159–170PubMedCrossRefGoogle Scholar
  19. 19.
    Vijayachandran LS, Viola C, Garzoni F et al (2011) Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells. J Struct Biol 175:198–208PubMedCrossRefGoogle Scholar
  20. 20.
    Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4:251–256PubMedCrossRefGoogle Scholar
  21. 21.
    Jeong JY, Yim HS, Ryu JY et al (2012) One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies. Appl Environ Microbiol 78:5440–5443PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Matthias Haffke
    • 1
  • Martin Marek
    • 2
  • Martin Pelosse
    • 1
  • Marie-Laure Diebold
    • 2
  • Uwe Schlattner
    • 3
  • Imre Berger
    • 1
  • Christophe Romier
    • 2
  1. 1.European Molecular Biology Laboratory (EMBL), Grenoble Outstation and Unit of Virus Host-Cell Interactions (UVHCI)Université Grenoble Alpes-EMBL-CNRS, UMR 5233Grenoble Cedex 9France
  2. 2.Département de Biologie Structurale Intégrative, Centre de Biologie IntégrativeInstitut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), UDS, CNRS, INSERMIllkirch CedexFrance
  3. 3.Laboratory of Fundamental and Applied Bioenergetics (LBFA)Université Grenoble Alpes, Inserm, U1055GrenobleFrance

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