Abstract
Toxic, membrane, and hydrophobic proteins are usually difficult to individually over-express in Escherichia coli because they require a binding-partner protein for folding and stability. To obtain these types of soluble proteins or protein complexes, protein co-expression is used. Such co-expression systems are extremely suitable for the high-throughput validation of protein–protein interactions. In a previous study, we developed a novel co-expression vector, pHEX, which is compatible, and thus can be partnered, with many commercially available E. coli vectors, such as pGEX and pMAL. Either of the vectors allows proteins to be expressed individually as a tagged fusion protein and can be used directly for protein co-purification. This protocol presents the experimental procedure for the co-expression method.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Makrides, S. C. (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 60, 512–538.
Wuu, J. J. and Swartz, J. R. (2008) High yield cell-free production of integral membrane proteins without refolding or detergents. Biochim. Biophys. Acta. 1778, 1237–1250.
Zhang, J., Zhang, Y., and Inouye, M. (2003) Characterization of the interactions within the mazEF addiction module of Escherichia coli. J. Biol. Chem. 278, 32300–32306.
Bross, P., Andresen, B. S., Winter, V., et al., (1993) Co-overexpression of bacterial GroESL chaperonins partly overcomes non-productive folding and tetramer assembly of E. coli-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) carrying the prevalent disease-causing K304E mutation. Biochim. Biophys. Acta. 1182, 264–274.
Romier, C., Ben Jelloul, M., 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, 1232–1242.
Held, D., Yaeger, K., and Novy, R. (2003) New co-expression vectors for expanded compatibilities in E.coli. inNovations 18, 4–6.
Wakamori, M., Umehara, T., and Yokoyama S. (2010) A series of bacterial co-expression vectors with rare-cutter recognition sequences. Protein Expr. Purif. 74, 88–98.
Scheich, C., Kümmel, D., Soumailakakis, D., et al., (2007) Vectors for co-expression of an unrestricted number of proteins. Nucleic Acids Res. 35, e43.
Dzivenu, O. K., Park, H. H., and Wu, H. (2004) General co-expression vectors for the overexpression of heterodimeric protein complexes in Escherichia coli. Protein Expr. Purif. 38, 1–8.
Stols, L., Zhou, M., Eschenfeldt, W. H., et al., (2007) New vectors for co-expression of proteins: structure of Bacillus subtilis ScoAB obtained by high-throughput protocols. Protein Expr. Purif. 53, 396–403.
Zeng, J., Zhang, L., Li, Y., et al., (2010) Over-producing soluble protein complex and validating protein-protein interaction through a new bacterial co-expression system. Protein Expr. Purif. 69, 47–53.
Wang, J., Jiang, P. X., Feng, H., et al., (2007) Three eukaryote-like Orc1/Cdc6 proteins functionally interact and mutually regulate their activities of binding to the replication origin in the hyperthermophilic archaeon Sulfolobus solfataricus P2. Biochem. Biophys. Res. Commun. 363, 63–70.
Zhang, L., Zhang, L., Liu, Y., et al., (2009) Archaeal eukaryote-like Orc1/Cdc6 initiators physically interact with DNA polymerase B1 and regulate its functions. Proc. Natl. Acad. Sci. USA 106, 7792–7797.
Sambrook. J., and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (30930003).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Zeng, J., He, ZG. (2012). A New Bacterial Co-expression System for Over-expressing Soluble Protein and Validating Protein–Protein Interaction. In: Lorence, A. (eds) Recombinant Gene Expression. Methods in Molecular Biology, vol 824. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-433-9_12
Download citation
DOI: https://doi.org/10.1007/978-1-61779-433-9_12
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-432-2
Online ISBN: 978-1-61779-433-9
eBook Packages: Springer Protocols