Tobacco etch virus protease

Stols, L., Gu, M., Dieckman, L., Raffen, R., CoUart, F. R. and Donnelly, M. 1. (2002). Anew vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. Protein Expr. Purif. 25, 8-15. 

GST, calmodulin-binding peptide, His-, FLAG-tag, protein A, glycoprotein D of HSV), or to enable its detection and/or selection, i.e. tags based on protein-reporters (/1-galactosidase, GFP, CAT, hGH) (Makrides, 1999). Tags can be fused to N- or C-terminal ends of the protein and a site for proteolytic cleavage is commonly included to eliminate the tag upon exploitation of its functionality. The proteases most commonly used are thrombin, enterokinase, factor Xa, and TEV (catalytic domain of Nia, the nuclear inclusion protein from tobacco etch virus). 

PS—alkyne probe, followed by a CC reaction to introduce a biotin tag with a tobacco etch virus (TEV) protease cleavage site. Tagged proteins were then enriched with streptavidin beads and subsequendy digested by trypsin. The supernatant contained peptide fragments of the enriched proteins and was, in contrast to the previous procedure, saved for subsequent analysis by multidimensional protein identification technology (MudPIT), a novel two-dimensional LC—MS/MS analysis method that will be discussed in more detail... 

As exemplified above, a ligation needs an N-terminal Cys and a C-terminal thioester as prerequisites. In chemical synthesis these two conditions are obviously easily achieved. However, recombinant proteins are synthesized with an N-terminal Met and the C-terminus is just a free carboxyl group. There are various methods available to generate an N-terminal Cys. In a commonly used strategy a specific protease cleaving site is introduced. The Tobacco Etch Virus (TEV) protease recognizes the sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) and cleaves the... 

The MK2 constructs (Table 1) were cloned using standard techniques and expressed in E. coli as glutathione S-transferase (GST) fusion proteins. The expression plasmids encoded GST followed by thrombin and tobacco etch virus (TEV) protease cleavage sites and the desired MK2 sequence. It proved important to... 

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