Structural Proteomics pp 63-89

Part of the Methods in Molecular Biology book series (MIMB, volume 1261)

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

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 

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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