| Product Description | Aladdin's TEV Protease is a Tobacco Etch Virus (TEV) cysteine protease recombinantly expressed in E. coli, with both Glutathione S-transferase and 6X Histidine tags. This product can specifically recognize the heptapeptide, Glu-Asn-Leu-Tyr-Phe-Gln-Gly/Ser, and cleave the peptide bond between Gln and Gly/Ser amino acid residues. It is often used for the removal of affinity purification tags such as GST or poly-histidine from fusion proteins.It is recommended to fuse GST or His tags at the N-terminus of target protein and add the heptapeptide sequence in between. After the removal of GST or His tag from fusion proteins by TEV protease, only one extra Gly/Ser amino acid residue is left at the N-terminus of the target protein, thereby minimizing the potential impact of the tag on the structure and function of target proteins. To express target proteins containing the TEV Protease recognition site, we recommend Aladdin's pET -N-His-TEV expression plasmid .The TEV Protease has an optimal working temperature of 30℃. It is active within the range of 29-34℃, but its enzymatic activity will drop sharply when the temperature reaches or exceeds 37℃. To preserve the structure and biological activity of the target protein, it is recommended to overnight digestion with TEV Protease at 4℃. TEV Protease is active between pH6.0 and 9.0. When the pH is close to or lower than 5, the enzymatic activity is lost completely.Another outstanding advantage of TEV Protease is that it remains active in 400mM imidazole. Therefore, TEV Protease can be directly added to the purified protein containing high concentrations of imidazole to cleave His tags. After cleavage, the excised tags and the TEV Protease can be removed by nickel column, while the target proteins with tags removed are eluted. For the purification of His-tagged proteins, we recommend using Aladdin's UltraBio™ His-tag Purification Resin (Reductant & Chelator-resistant) , His-tag Protein Purification Kit (Reductant & Chelator-resistant) , UltraBio™ His-tag Purification Resin (Denaturant-resistant) , or His-tag Protein Purification Kit (Denaturant-resistant) .The enzyme activity of TEV Protease is not inhibited by common serine protease inhibitors such as PMSF, AEBSF, bestatin, pepstatin, E-64, TLCK, and EDTA. However, protease inhibitors targeting cysteine residues, such as NEM or IAA, can significantly inhibit the TEV Protease activity because the TEV Protease contains the Cys151 which is essential for its enzyme activity.Compared with TEV Protease (His-tag) , the biggest advantage of TEV Protease is that it is suitable for the removal of both His and GST tags from fusion proteins with the TEV Protease cleavage sites. After cleavage, undigested proteins, excised tags and TEV Protease can be removed by affinity chromatography on a nickel-chelating resin or GST purification resin, while the target proteins with tags removed will be in the flow-through fractions.Unit Definition
Enzyme storage buffer： 25mM Tris-HCl, 150mM NaCl, 1mM EDTA, 5mM DTT, 50%(v/v) glycerol, pH 8.0.10X TEV Buffer: 500mM Tris-HCl, 500mM NaCl, 5mM EDTA, 10mM DTT, pH 8.0.Per ml of this product contains 1000 units of enzyme, which is sufficient for cleaving 3mg of fusion proteins with TEV Protease recognition site.
Precautions： This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use： 1. Optimization of cleavage conditions. The ratio of TEV Protease to tagged protein should be optimized for a particular protein as follows:a. Reaction setupComponentVolumeH2OX10X TEV Buffer5Tag protein (8μg)YTEV Protease (1U/μl)0, 1.5 or 2.5TotalNote: If the concentration of tagged protein is 2μg/μl, then Y=8/2=4μl.B. Incubate the reaction mixture at 30℃ for 1, 2, 4, or 6 hours. If the target protein is heat-labile, incubate at 4℃ overnight . Generally, the tags can be completely excised and removed after 1 h at 30℃ or after 16 h at 4℃.C. Analyze 20μl of the reaction product by SDS-PAGE to determine the optimal enzyme amount and incubation time required for a particular fusion protein.
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