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| Catalog | Product Name | EC No. | CAS No. | Source | Price |
|---|---|---|---|---|---|
| NATE-0922 | Tobacco Etch Virus Protease, Recombinant | E. coli | Inquiry |
TEV Protease is a highly specific cysteine protease that recognizes the amino-acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) and cleaves between the Gln and Gly/Ser residues. TEV Protease, also known as Tobacco Etch Virus (TEV) Protease, is a highly specific cysteine protease that recognizes the amino-acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) and cleaves between the Gln and Gly/Ser residues. It is often used for the removal of affinity purification tags such as maltose-binding protein (MBP) or poly-histidine from fusion proteins. TEV Protease has a 7xHis-tag for easy removal from a reaction using nickel affinity resins and has been engineered to improve thermal stability and decrease autolysis.
At present, X-ray crystallography has solved the structure of TEV protease. It consists of two β barrels and a flexible C-terminal tail, and has structural homology with the chymotrypsin superfamily of proteases (PA family, C4 family classified by MEROPS). Although TEV protease is homologous to cellular serine proteases (such as trypsin, elastase, thrombin, etc.), it uses cysteine as its catalytic nucleophile (as are many other viral proteases). Covalent catalysis is carried out with an Asp-His-Cys triplet, separated between two barrels (Asp on β1 and His and Cys on β2). The matrix is kept in the shape of a beta sheet, forming an anti-parallel interaction with the cracks between the barrels and a parallel interaction with the tail end of the C-terminal. Therefore, the enzyme forms a binding channel around the substrate, and side chain interactions control specificity.
Figure 1. Structure of TEV Protease.
Tobacco etching virus encodes its entire genome as a single large multi-protein (350 kDa). It is cleaved into functional units by three proteases: P1 protease (1 cleavage site), auxiliary component protease (1 cleavage site) and TEV protease (7 cleavage sites). The natural TEV protease also contains internal self-cleavage sites. Slowly cutting this site inactivates the enzyme (physiological reasons are not clear).
One of the main purposes of this protein is to remove affinity tags from purified recombinant fusion proteins. The reason for using TEV protease as a biochemical tool is its high sequence specificity. When the priority sequence is inserted into the flexible loop, this specificity allows controlled cleavage of the protein. Since there are almost no recognized sequences in the protein, it also makes it relatively non-toxic in the body. Although rational design has had limited success in changing protease specificity, directed evolution has been used to change the preferred residues before or after or after the cleavage site. However, TEV protease does have limitations as a biochemical tool. It is easily inactivated by self-cleavage (autolysis), although it can be eliminated by a single S219V mutation at the internal cleavage site. The protease expressed alone is also insoluble, but some attempts have been made to improve its solubility through directed evolution and computational design. It has also been shown that expression can be improved by fusion with maltose binding protein (MBP), which acts as a solubility enhancing partner. The molecular weight of the enzyme varies between 25 and 27 kDa, depending on the specific construct used.
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