One unit is defined as the amount of enzyme needed to cleave 3 μg of fusion protein in 1 hour to 85 % completion at 30°C in a buffer containing 50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, and 1 mM DTT.
Alternative Names
TEV; TEV Protease
Purity
> 90 % by SDS-PAGE analysis
Format
Liquid
Buffer
Sterile-filtered 25 mM Tris-HCl, pH 8.0, 0.25 mM EDTA, 0.5 mM DTT, containing 50% Glycerol
Preservative
None
Storage
Store as supplied at -20°C to -80°C for up to 6 months. Once opened under sterile conditions, store at -20°C to -80°C for up to 3 months. Avoid repeated freeze-thaw cycles.
Introduction
TEV protease encoded by the tobacco etch virus is a catalytic domain of the Nuclear Inclusion a (NIa) protein. It consists of 241 aa with the molecular weight of 27 kDa. TEV recognizes the amino acid sequence of the general form E-X-X-Y-X-Q (or S)/X’, and cleaves between Q (or S)/X’. In this form X and X’ stand for any of the amino acid residues, except that X’ cannot be P. The optimal cleavage site is ENLYFQ/G. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The mutants, S219V, was not only far more stable than the wild-type protease (~100-fold), but also a more efficient catalyst. As having the absolute specificity and wildly using conditions like broad pH range and ionic strength, the TEV protease became more versatile than EK, thrombin and other protease used in biochemical applications, especially recombinant protein production. The optimal temperature for cleavage is 30°C; however, the enzyme can be used at temperatures as low as 4°C. Following digestion, TEV Protease can be removed from the reaction via the GST tag sequence by GST chromatography.
Keywords
TEV; TEV Protease
Citations
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Background
TEV protease is a 27 kDa enzyme found in its native form as the C-terminal portion of the TEV polyprotein. It cleaves proteins at a specific recognition sequence of seven amino acids: Glu-Asn-Leu-Tyr-Phe-Gln-Gly/Ser (E-N-L-Y-F-Q-G/S), with cleavage occurring between Glu and Gly/Ser. This strict specificity makes TEV protease useful for cleaving fusion proteins where this sequence is present between the carrier domain and the protein of interest. Native TEV protease is not effectively inhibited by common serine protease inhibitors but can be inhibited by cysteine-targeting inhibitors that target cysteine residues, such as NEM and IAA. It is most active at temperatures between 29-34°C and pH 6-9, losing activity at higher temperatures and acidic conditions. While its solubility in aqueous media is limited, using protein tags such as GST and His-tag improves its solubility and allows for easier purification.
TEV protease has found widespread utility as a biochemical tool, particularly in the controlled cleavage of fusion proteins and the removal of affinity tags. Its high sequence specificity allows for precise cleavage at desired sites when the preferred sequence is inserted into flexible loops. However, autolysis or self-cleavage of TEV protease can lead to enzyme deactivation, requiring strategies to mitigate this issue. We have constructed a TEV protease mutant that is highly resistant to autolysis. S219V mutant of TEV protease does not undergo autolysis under any conditions but is twofold less active than the wild-type enzyme. Moreover, the recombinant TEV Protease (S219V) [GST] is a recombinant version of TEV protease containing a GST tag, facilitating its purification through chromatography.
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References
A combined approach to improving large-scale production of tobacco etch virus protease
Tobacco etch virus NIa proteinase (TEV protease) is an important tool for the removal of fusion tags from recombinant proteins. Production of TEV protease in Escherichia coli has been hampered by insolubility and addressed by many different strategies. However, the best previous results and newer approaches for protein expression have not been combined to test whether further improvements are possible. Here, we use a quantitative, high-throughput assay for TEV protease activity in cell lysates to evaluate the efficacy of combining several previous modifications with new expression hosts and induction methods. Small-scale screening, purification and mass spectral analysis showed that TEV protease with a C-terminal poly-Arg tag was proteolysed in the cell to remove four of the five arginine residues. The truncated form was active and soluble but in contrast, the tagged version was also active but considerably less soluble. An engineered TEV protease lacking the C-terminal residues 238–242 was then used for further expression optimization. From this work, expression of TEV protease at high levels and with high solubility was obtained by using auto-induction medium at 37 °C. In combination with the expression work, an automated two-step purification protocol was developed that yielded His-tagged TEV protease with >99% purity, high catalytic activity and purified yields of ～400 mg/L of expression culture (～15 mg pure TEV protease per gram of E. coli cell paste). Methods for producing glutathione-S-transferase-tagged TEV with similar yields (～12 mg pure protease fusion per gram of E. coli cell paste) are also reported.
Structural basis for the substrate specificity of tobacco etch virus protease
Because of its stringent sequence specificity, the 3C-type protease from tobacco etch virus (TEV) is frequently used to remove affinity tags from recombinant proteins. It is unclear, however, exactly how TEV protease recognizes its substrates with such high selectivity. The crystal structures of two TEV protease mutants, inactive C151A and autolysis-resistant S219D, have now been solved at 2.2- and 1.8-Å resolution as complexes with a substrate and product peptide, respectively. The enzyme does not appear to have been perturbed by the mutations in either structure, and the modes of binding of the product and substrate are virtually identical. Analysis of the protein-ligand interactions helps to delineate the structural determinants of substrate specificity and provides guidance for reengineering the enzyme to further improve its utility for biotechnological applications.
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