Characterization and Production of Protein Complexes by Co-expression in Escherichia coli
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 wordsProtein complexes Co-expression Escherichia coli ACEMBL pET-MCN pET-MCP Cloning SLIC Plasmid concatenation Expression tests High throughput Automation
The authors are supported by institutional funds from the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), the Université de Strasbourg (UDS), the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05-01), and Instruct, part of the European Strategy Forum of Research Infrastructures (ESFRI), and supported by national member subscriptions. IB acknowledges support from the European Commission (EC) Framework Programme (FP) 7 ComplexINC project (Contract Nr. 279039).
- 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
- 10.Held D, Yaeger K, Novy R (2003) New coexpression vectors for expanded compatibilities in E. coli. inNovations 18:4–6Google Scholar
- 11.Novy R, Yaeger K, Held D, Mierendorf R (2002) Coexpression of multiple target proteins in E. coli. inNovations 15:2–6Google Scholar
- 13.Selleck W, Tan S (2008) Recombinant protein complex expression in E. coli. Curr Protoc Protein Sci. Chapter 5, Unit 5 21Google Scholar