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.
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Acknowledgments
This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
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Raran-Kurussi, S., Cherry, S., Zhang, D., Waugh, D.S. (2017). Removal of Affinity Tags with TEV Protease. In: Burgess-Brown, N. (eds) Heterologous Gene Expression in E.coli. Methods in Molecular Biology, vol 1586. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6887-9_14
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DOI: https://doi.org/10.1007/978-1-4939-6887-9_14
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6885-5
Online ISBN: 978-1-4939-6887-9
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