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.
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| http://dx.doi.org/10.1007/978-1-4939-6887-9_14 | DOI Listing |
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