Protocol

Structural Proteomics

Volume 1261 of the series Methods in Molecular Biology pp 63-89

Date:

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

  • Matthias HaffkeAffiliated withEuropean Molecular Biology Laboratory (EMBL), Grenoble Outstation and Unit of Virus Host-Cell Interactions (UVHCI), Université Grenoble Alpes-EMBL-CNRS, UMR 5233 
  • , Martin MarekAffiliated withDépartement de Biologie Structurale Intégrative, Centre de Biologie Intégrative, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), UDS, CNRS, INSERM 
  • , Martin PelosseAffiliated withEuropean Molecular Biology Laboratory (EMBL), Grenoble Outstation and Unit of Virus Host-Cell Interactions (UVHCI), Université Grenoble Alpes-EMBL-CNRS, UMR 5233
  • , Marie-Laure DieboldAffiliated withDépartement de Biologie Structurale Intégrative, Centre de Biologie Intégrative, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), UDS, CNRS, INSERM
  • , Uwe SchlattnerAffiliated withLaboratory of Fundamental and Applied Bioenergetics (LBFA), Université Grenoble Alpes, Inserm, U1055
  • , Imre BergerAffiliated withEuropean Molecular Biology Laboratory (EMBL), Grenoble Outstation and Unit of Virus Host-Cell Interactions (UVHCI), Université Grenoble Alpes-EMBL-CNRS, UMR 5233
  • , Christophe RomierAffiliated withDépartement de Biologie Structurale Intégrative, Centre de Biologie Intégrative, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), UDS, CNRS, INSERM Email author 

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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