Expression and Purification of Recombinant Proteins in Escherichia coli with a His6 or Dual His6-MBP Tag
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 wordsFusion 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.
- 9.Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar