Summary
This chapter describes a simple method for overproducing a soluble form of the tobacco etch virus (TEV) protease in Escherichia coli and purifying it to homogeneity so that it may be used as a reagent for removing affinity tags from recombinant proteins by site-specific endoproteolysis. The protease is initially produced as a fusion to the C-terminus of E. coli maltose binding protein (MBP), which causes it to accumulate in a soluble and active form rather than in inclusion bodies. The fusion protein subsequently cleaves itself in vivo to remove the MBP moiety, yielding a soluble TEV protease catalytic domain with an N-terminal polyhistidine tag. The His-tagged TEV protease can be purified in two steps using immobilized metal affinity chromatography (IMAC) followed by gel filtration. An S219V mutation in the protease reduces its rate of autolysis by approximately 100-fold and also gives rise to an enzyme with greater catalytic efficiency than the wild-type protease.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Waugh, D. S. (2005) Making the most of affinity tags.Trends Biotechnol 23, 316–320.
Goel, A., Beresford, G. W., Colcher, D., Pavlinkova, G., Booth, B. J., Baranowska-Kortylewicz, J., and Batra, S. K. (2000) Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct.Biochim Biophys Acta 1523, 13–20.
Bucher, M. H., Evdokimov, A. G., and Waugh, D. S. (2002) Differential effects of short affinity tags on the crystallization ofPyrococcus furiosus maltodextrin-binding protein.Acta Crystallogr D Biol Crystallogr 58, 392–397.
Fonda, I., Kenig, M., Gaberc-Porekar, V., Pristovaek, P., and Menart, V. (2002) Attachment of histidine tags to recombinant tumor necrosis factor-alpha drastically changes its properties.Sci World J 2, 1312–1325.
Chant, A., Kraemer-Pecore, C. M., Watkin, R., and Kneale, G. G. (2005) Attachment of histidine tag to the minimal zinc finger protein of theAspergillus nidulans gene regulatory proteiin AreA causes a confor-mational change at the DNA-binding site.Protein Expr Purif 39, 152–159.
Arnau, J., Lauritzen, C., Petersen, G. E., and Pedersen, J. (2006) Current strategies for the use of affinity tags and removal for the purification of recombinant proteins.Protein Expr Purif 48, 1–13.
Choi, S. I., Song, H. W., Moon, J. W., and Seong, B. L. (2001) Recombinant enterok-inase light chain with affinity tag: expression from Saccharomyces cerevisiae and its utilities in fusion protein technology.Biotechnol Bioeng 75, 718–724.
Jenny, R. J., Mann, K. G., and Lundblad, R. L. (2003) A critical review of the methods for cleavage of fusion proteins with thrombin and factor Xa.Protein Expr Purif 31, 1–11.
Parks, T. D., Leuther, K. K., Howard, E. D., Johnston, S. A., and Dougherty, W. G. (1994) Release of proteins and peptides from fusion proteins using a recombinant plant virus protease.Anal Biochem 216, 413–417.
Kapust, R. B., Tozser, J., Copeland, T. D., and Waugh, D. S. (2002) The P1'′ specificity of tobacco etch virus protease.Biochem Biophys Res Commun 294, 949–955.
Kapust, R. B., and Waugh, D. S. (1999)Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused.Protein Sci 8, 1668–1674.
Lucast, L. J., Batey, R. T., and Doudna, J. A. (2001) Large-scale purification of a stable form of recombinant tobacco etch virus protease.Biotechniques 30, 544–546.
Parks, T. D., Howard, E. D., Wolpert, T. J., Arp, D. J., and Dougherty, W. G. (1995) Expression and purification of a recom-binant tobacco etch virus NIa proteinase: biochemical analyses of the full-length and a naturally occurring truncated proteinase form.Virology 210, 194–201.
Kapust, R. B., Tozser, J., Fox, J. D., Anderson, D. E., Cherry, S., Copeland, T., and Waugh, D. S. (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic efficiency.Protein Eng 14, 993–1000.
Tropea, J. E., Cherry, S., Nallamsetty, S., Bignon, C., and Waugh, D. S. (2007) A generic method for the production of recombinant proteins inEscherichia coli using a dual hexahistidine-maltose-binding protein affinity tag.Methods Mol Biol 363, 1–19.
Mohanty, A. K., Simmons, C. R., and Wiener, M. C. (2003) Inhibition of tobacco etch virus protease activity by detergents.Protein Expr Purif 27, 109–114.
Nallamsetty, S., Kapust, R. B., Tozser, J., Cherry, S., Tropea, J. E., Copeland, T. D., and Waugh, D. S. (2004) Efficient site-specific cleavage of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro.Protein Expr Purif 38, 108–115.
Acknowledgments
This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science + Business Media
About this protocol
Cite this protocol
Tropea, J.E., Cherry, S., Waugh, D.S. (2009). Expression and Purification of Soluble His6-Tagged TEV Protease. In: Doyle, S.A. (eds) High Throughput Protein Expression and Purification. Methods in Molecular Biology, vol 498. Humana Press. https://doi.org/10.1007/978-1-59745-196-3_19
Download citation
DOI: https://doi.org/10.1007/978-1-59745-196-3_19
Publisher Name: Humana Press
Print ISBN: 978-1-58829-879-9
Online ISBN: 978-1-59745-196-3
eBook Packages: Springer Protocols