Rescuing Aggregation-Prone Proteins in Escherichia coli with a Dual His6-MBP Tag

  • Danielle Needle
  • David S. WaughEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1177)


Insolubility of recombinant proteins in Escherichia coli is a major impediment to their production for structural and functional studies. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely recognized as a premier solubilizing agent. In this chapter, we describe how to construct dual His6-MBP-tagged fusion proteins by Gateway® recombinational cloning and how to predict their yield and solubility. We also describe a simple and rapid procedure to test the ability of a His6-MBP fusion protein to bind to Ni-NTA resin and to be digested by tobacco etch virus (TEV) protease, along with a method to assess the solubility of the target protein after it has been separated from His6-MBP.

Key words

Maltose-binding protein MBP Fusion protein Solubility enhancer TEV protease Tobacco etch virus protease Hexahistidine tag His-tag His6-MBP Gateway® cloning Recombinational cloning 



We thank Di Zhang for constructing the His6-MBP-DHFR expression vector. This research was funded by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the US Government.


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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Macromolecular Crystallography Laboratory, Center for Cancer ResearchNational Cancer InstituteFrederickUSA

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