Abstract
In many cases, the analysis of a specific protein is impeded by the inability to purify large amounts of it from a native source. Proteins of interest may be present in minute quantities and/or purification may be plagued with technical problems. Recombinant DNA methodologies have enabled researchers to circumvent some of these limitations by producing and purifying large quantities of protein in a nonnative system. Various systems and strategies have been successfully employed, depending on the specific protein of interest and the desired use of the final end product (antibody production, crystallography studies etc.). This chapter reviews some common methods for the production of recombinant fusion proteins and specifically describes a versatrle method for the removal of affinity tags from recombinant fusions using a highly purified proteinase with an unparalleled degree of specificity. This proteinase, from the genome of tobacco etch virus (TEV), demonstrates specific proteolytic activity under a wide range of parameters (salt, temperature, pH), making it an excellent choice for cleavage of fusion proteins (1,2).
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
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 proteinase. Anal Biochem. 216, 413–417
Parks T D., Howard E. D, Wolpert T. J., Arp D J., and Dougherty W G (1995) Expression and purification of a recombinant tobacco etch virus NIa proteinase biochemical analyses of the full length and a naturally occurring truncated proteinase form. Virology 210, 194–201.
Goeddel D. V (1990) Systems for heterologous gene expression Methods Enzymol 185, 3–7
Studier F. W, Rosenberg A H., Dunn J. J, and Dubendorff J J (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185, 60–69
deBoer H A., Comstock L J., and Vasser M (1983) The tac promoter. a functional hybrid derived from trp and lac promoters. Proc Natl Acad Sci USA 80, 21–25
Brosius J, Erfle M, and Storella J (1985) Spacing of the -10 and -35 regions in the tac promoter Effect on its in vivo activity J Biol Chem. 260, 3539–3541.
Miller L. K (1988) Baculoviruses as gene expression vectors. Ann Rev Microbiol. 42, 177–199
Uhlen M. and Moks T (1990) Gene fusions for purpose of expression an introduction Methods Enzymol 185, 129–143.
Smith D. B and Johnson K. S (1988) Single step purification of polypeptides expressed in Eschericliza coli as fusions with glutathione-S-transferase. Gene 67, 31–40
Nilsson B. and Abrahmsen L (1990) Fusions to Staphylococcal protein A Methods Enzymol 185, 144–161.
Hochuli E., Doebeli H., and Schacher A. (1987) New metal chelate absorbant selective for proteins and peptides containg neighboring histidine residues J Chromatogr 411, 177–184
Carter P (1990) Site-specific proteolysls of fusion proteins, in Protein Purification. American Chemical Society, Washington, DC, pp 181–193.
Maroux S., Baratti J., and Desnuelle P (1971) Purification and specificity of procine enterokinase J Biol. Chem 246, 5031–5039.
Dougherty W. G and Semler B L. (1993) Expression of virus-encoded proteinases. functional and structural similarities with cellular enzymes. Micro Rev 57, 781–822
Carrington J. C. and Dougherty W G (1987) Small nuclear inclusion protein encoded by a plant potyvirus genome is a protease. J. Virol 61, 2540–2548.
Dougherty W G, Parks T. D., Cary S. M., Bazan J F, and Flettenck R J (1989) Characterization of the catalytic residues of the tobacco etch virus 49-kDa proteinase. Virology 172, 302–310
Carrington J. C and Dougherty W. G (1988) A viral cleavage site cassette. ldentification of amino acid sequences required for tobacco etch virus polyprotem processing Proc Natl Acad Sci USA 85, 3391–3395
Dougherty W. G., Cary S M, and Parks T. D (1989) Molecular genetic analysis of a plant vrius polyprotem cleavage site a model Virology 171, 356–364
Dougherty W G. and Parks T D. (1989) Molecular genetic and biochemical evidence for the involvement of the heptapeptide cleavage sequence in determing the reaction profile at two tobacco etch virus cleavage sites in cell-free assays. Virology 172, 145–155
Parks T D. and Dougherty W. G. (1991) Substrate recogition by the NIa proteinase of two potyviruses involves multiple domains. characterization using genetically engineered hybrid molecules Virology 182, 17–27
Polayes D. A., Goldstein A, Ward G., and Hughes A J, Jr (1994) TEV protease, recombinant. a site-specific protease for efficient cleavage of affinity tags from expressed proteins. FOCUS 16, 2–5
Polayes D (1996) Prokaryotic protein expression and purification with the PROEX™ expression system FOCUS 18, 50–53
Sambrook J., Fritsch E. F, and Manitis T. (1989) Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Laemmli U K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (Lond) 227, 680–685.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1998 Humana Press Inc.
About this protocol
Cite this protocol
Polayes, D.A., Parks, T.D., Johnston, S.A., Dougherty, W.G. (1998). Application of TEV Protease in Protein Production. In: Reischl, U. (eds) Molecular Diagnosis of Infectious Diseases. Methods in Molecular Medicine™, vol 13. Humana Press, Totowa, NJ. https://doi.org/10.1385/0-89603-485-2:169
Download citation
DOI: https://doi.org/10.1385/0-89603-485-2:169
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-485-3
Online ISBN: 978-1-59259-597-6
eBook Packages: Springer Protocols