Abstract
Maltose-binding protein (MBP) is one of the most popular fusion partners being used for producing recombinant proteins in bacterial cells. MBP allows one to use a simple capture affinity step on amylose–agarose columns, resulting in a protein that is often 70–90% pure. In addition to protein-isolation applications, MBP provides a high degree of translation and facilitates the proper folding and solubility of the target protein. This chapter describes efficient procedures for isolating highly purified MBP-target proteins. Special attention is given to considerations for downstream applications such as structural determination studies, protein activity assays, and assessing the chemical characteristics of the target protein.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Nikaido, H. (1994) Maltose transport system of Escherichia coli: an ABC-type transporter. FEBS Lett. 346, 55–58.
Baneyx, F., and Mujacic, M. (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat. Biotech. 22, 1399–1408.
Randall, L.L., Topping, T.B., Smith, V.F., Diamond, D.L., and Hardy, S.J. (1998). SecB: A chaperone from Escherichia coli. Meth. Enzymol. 290, 444–459.
Nomine, Y., Ristriani, T., Laurent, C., Lefevre, J.F., Weiss, E., and Trave, G. (2001).A strategy for optimizing the monodispersity of fusion proteins: application to purification of recombinant HPV E6 oncoprotein. Prot. Eng. 14, 297–305.
Sachdev, D., and Chirgwin, J.M. (1998). Order of fusions between bacterial and mammalian proteins can determine solubility in Escherichia coli. Biochem. Biophys. Res. Comm. 244, 933–937.
Nallamsetty, S. and Waugh, D.S. (2006) Solubility-enhancing proteins MBP and NusA play a passive role in the folding of their fusion partners. Protein Expr. Purif. 45, 175–182.
Lee, R.T., Ichikawa, Y., Allen, H.J., and Lee, Y.C. (1990) Binding characteristics of galactoside-binding lectin (galaptin) from human spleen. J. Biol. Chem. 265, 7864–7871.
Kapust, R.B. and Waugh, D.S. (2000). Controlled intracellular processing of fusion proteins by TEV protease. Protein Expr Purif. 19, 312–318
Nallamsetty, S., and Waugh, D.S. (2007) A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag. Nat. Protoc. 2, 383–391.
Mohanty, A., Simmons, C.R., and Wiener, M.C. (2003) Inhibition of tobacco etch virus protease activity by detergents. Protein Expr. Purif. 27, 109–114.
Sheffield, P., Garrard, S., and Derewenda, Z. (1999) Overcoming expression and purification problems of RhoGDI using a family of “parallel” expression vectors. Protein Expr. Purif. 15(1), 34–39.
Kapust, R.B., Tözsér, J., Fox. J.D., Anderson, D.E., Cherry, S., Copeland, T.D., and Waugh, D.S. (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic efficiency. Protein Eng. 14, 993–1000.
Riggs, P. (2000) Expression and purification of recombinant proteins by fusion to maltose-binding protein. Mol. Biotechnol. 15, 51–63.
Nallamsetty, S., Austin, B.P., Penrose, K.J. and Waugh, D.S. (2005) Gateway vectors for the production of combinatorially-tagged His6-MBP fusion proteins in the cytoplasm and periplasm of Escherichia coli. Protein Sci. 14, 2964–2971.
Smith, D. (2003) Crystal structures of fusion proteins with large-affinity tags. Protein Sci. 12, 1313–1322.
Tropea, J.E., Cherry, S., Nallamsetty, S., Bignon, C., and Waugh, D.S. (2007) A generic method for the production of recombinant proteins in Escherichia coli using a dual His6-MBP affinity tag. Methods Mol. Biol. 363, 1–19.
Austin, B.P., Nallamsetty, S., and Waugh DS (2009) Hexahistidine-tagged maltose-binding protein as a fusion partner for the production of soluble recombinant proteins in Escherichia coli. Methods Mol. Biol. 498, 157–172.
Pattenden, L.K., and Thomas, W.G. (2008) Amylose affinity chromatography of maltose-binding protein: purification by both native and novel matrix-assisted dialysis refolding methods. Methods Mol. Biol. 421, 169–189.
Acknowledgments
We thank Dr. Erez Podoly from the Biological Chemistry Department of The Hebrew University of Jerusalem, now at the Structural Biology Department of Stanford University School of Medicine, for expressing the fusion protein, for his assistance with purification and his helpful suggestions. We thank Dr. Daria Mochly-Rosen from the Department of Chemical and Systems Biology, School of Medicine, Stanford University for kindly providing us with pMAL-c2MBP-Rack1.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Lebendiker, M., Danieli, T. (2011). Purification of Proteins Fused to Maltose-Binding Protein. In: Walls, D., Loughran, S. (eds) Protein Chromatography. Methods in Molecular Biology, vol 681. Humana Press. https://doi.org/10.1007/978-1-60761-913-0_15
Download citation
DOI: https://doi.org/10.1007/978-1-60761-913-0_15
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-912-3
Online ISBN: 978-1-60761-913-0
eBook Packages: Springer Protocols