Tobacco etch virus

The plasmid vector pTEV-DHFR was used in this study. Internal ribosome entry sequence originated from tobacco etch virus (TEV) was locatai at upstream of dihydrofolate reductase (DHFR) gene. 

Granulocyte-macrophage Suspension Nicotiana tabacum A. tumefaciens CaMV 35 S Tobacco etch virus 150 UgL"1 (i) 40... 

Moyer, J. W., and S. H. Smith. Oxidant injury reduction on tobacco induced by tobacco etch virus infection. Environ. Pollut. 9 103-106, 1975. 

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. 

Fig. 5 Tandem orthogonal proteolysis (TOP) ABPP. TEV Tobacco etch virus...

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... 

As a proof-of-principle, this method has been used to generate a dual-labeled Rab7 GTPase. To generate Rab7 with an N-terminal cysteine, a tobacco etch virus... 

Fig, 1, pET-derived expression vectors harboring various fusion partners, (a) Schematic representation of the expression cassette, (b) List of the vectors, fusion tags, and their targeted cellular localization. MBPmaltose-binding protein, GB1 pi -domain of the Streptococcal protein G, Trx Thioredoxin, DsbA Protein Disulfide Isomerase A, DsbC Protein Disulfide Isomerase C, GST Glutathione S-transferase, 7 l/Tobacco Etch Virus. For the DsbC and DsbA fusions, two versions were constructed, for secretion Into the periplasm and for intracellular expression. 

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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