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.
Affinity chromatography Affinity tag Fusion protein His-tag IMAC Immobilized metal affinity chromatography Maltose-binding protein MBP TEV protease Tobacco etch virus protease
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This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
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