Removal of Affinity Tags with TEV Protease

  • Sreejith Raran-Kurussi
  • Scott Cherry
  • Di Zhang
  • David S. Waugh
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1586)

Abstract

Although affinity tags are highly effective tools for the expression and purification of recombinant proteins, they generally need to be removed prior to structural and functional studies. This chapter describes a simple method for overproducing a soluble form of a stable variant of tobacco etch virus (TEV) protease in Escherichia coli and a protocol for purifying it to homogeneity so that it can be used as a reagent for removing affinity tags from recombinant proteins by site-specific endoproteolysis. Further, we cleave a model substrate protein (MBP-NusG) in vitro using the purified TEV protease to illustrate a protease cleavage protocol that can be employed for simple pilot experiments and large-scale protein preparations.

Key words

Affinity chromatography Affinity tag Fusion protein His-tag IMAC Immobilized metal affinity chromatography Maltose-binding protein MBP TEV protease Tobacco etch virus protease 

Notes

Acknowledgments

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Sreejith Raran-Kurussi
    • 1
  • Scott Cherry
    • 1
  • Di Zhang
    • 1
  • David S. Waugh
    • 1
  1. 1.Macromolecular Crystallography Laboratory, Center for Cancer ResearchNational Cancer Institute at FrederickFrederickUSA

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