Expression and Purification of Recombinant Proteins in Escherichia coli with a His6 or Dual His6-MBP Tag

  • Sreejith Raran-Kurussi
  • David S. WaughEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1607)


Rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in Escherichia coli. Affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. Yet insolubility of recombinant proteins in E. coli remains a persistent problem. 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 acknowledged as a highly effective solubilizing agent. In this chapter, we describe how to construct either a His6- or a dual His6-MBP tagged fusion protein by Gateway® recombinational cloning and how to evaluate their yield and solubility. We also describe a simple and rapid procedure to test the solubility of proteins after removing their N-terminal fusion tags by tobacco etch virus (TEV) protease digestion. The choice of whether to use a His6 tag or a His6-MBP tag can be made on the basis of this solubility test.

Key words

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



We thank Karina Keefe and Danielle Needle for constructing the ChikV protease and MERS-CoV 3CLproC148A expression vectors, respectively. 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.


  1. 1.
    Waugh DS (2005) Making the most of affinity tags. Trends Biotechnol 23:316–320CrossRefPubMedGoogle Scholar
  2. 2.
    Kapust RB, Waugh DS (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8:1668–1674CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Fox JD, Routzahn KM, Bucher MH et al (2003) Maltodextrin-binding proteins from diverse bacteria and archaea are potent solubility enhancers. FEBS Lett 537:53–57CrossRefPubMedGoogle Scholar
  4. 4.
    Tropea JE, Cherry S, Nallamsetty S et al (2007) A generic method for the production of recombinant proteins in Escherichia coli using a dual hexahistidine-maltose-binding protein affinity tag. Methods Mol Biol 363:1–19CrossRefPubMedGoogle Scholar
  5. 5.
    Routzahn KM, Waugh DS (2002) Differential effects of supplementary affinity tags on the solubility of MBP fusion proteins. J Struct Funct Genom 2:83–92CrossRefGoogle Scholar
  6. 6.
    Kapust RB, Tozser J, Fox JD et al (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic efficiency. Protein Eng 14:993–1000CrossRefPubMedGoogle Scholar
  7. 7.
    Fox JD, Waugh DS (2003) Maltose-binding protein as a solubility enhancer. Methods Mol Biol 205:99–117PubMedGoogle Scholar
  8. 8.
    Brody JR, Kern SE (2004) Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. BioTechniques 36:214–216PubMedGoogle Scholar
  9. 9.
    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Macromolecular Crystallography Laboratory, Center for Cancer ResearchNational Cancer Institute at FrederickFrederickUSA

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