TEV 蛋白酶 (His+GST 标签) 是一种在大肠杆菌中重组表达的带 His、GST 双标签的来源于烟草蚀纹病毒 (TEV) 的半胱氨酸蛋白酶，能特异性地识别七肽序列 Glu-Asn-Leu-Tyr-Phe-Gln-Gly/Ser，并在 Gln 和 Gly/Ser 氨基酸残基之间进行酶切，常用于去除融合蛋白的 GST、His 或者其它标签的蛋白酶。TEV 蛋白酶 (His+GST 标签) 酶切上述七肽序列，标签蛋白被酶切后，在目的蛋白的 N 端仅有一个额外的 Gly/Ser 氨基酸残基，最大限度地减少了对目的蛋白结构和功能的影响。TEV 蛋白酶另一显著优点是在 400mM 咪唑中仍有较高活力，因此对于很多用镍柱纯化的带 His 或 GST 标签目的蛋白，可直接将 TEV 蛋白酶加入含高浓度咪唑的蛋白溶液中，在 4℃透析去除咪唑的同时进行酶切。也可以在透析后再用 TEV 蛋白酶进行酶切以去除标签。
TEV 蛋白酶 (His+GST 标签) 适用于酶切带有 His 或 GST 标签的重组蛋白，因为酶切后没有完全酶切的重组蛋白、切除的 His 标签、GST 标签以及 TEV 蛋白酶 (His+GST 标签) 都可以结合于 His 或 GST 纯化介质上而被除去，穿柱液中则含有所需的靶蛋白。
用 途
生物工程下游技术中融合蛋白酶切使用的重要工具酶
可用于 TEV 蛋白酶识别位点的融合蛋白的切割。
推荐酶切条件：
| 成分 | 体积 |
|---|
| 融合蛋白 | 0.1mg | | TEV 蛋白酶 | 20U | | 10× TEV buffer | 10μl | | H₂O | 补加至 100μl | | 温度（℃） | 20-37℃ | | 时间（h） | 12~16 |
|
使用注意事项：
1）TEV 蛋白酶的最佳酶切温度是 30℃，在 29-34℃范围内均具有较高的酶活性，当温度达到或高于 37℃时，其酶活性会急剧下降。在实际操作过程中，为尽量保留目的蛋白的结构和生物活性，建议在 4℃用 TEV 蛋白酶切过夜。TEV 蛋白酶在 pH6.0-9.0 范围内具有活性，而当 pH 小于或等于 5 时，会失去酶活性。
2）TEV 蛋白酶的活性不会被常见的丝氨酸蛋白酶抑制剂如 PMSF、AEBSF、Bestatin、Pepstatin、E-64、TLCK 和 EDTA 所抑制。但靶向半胱氨酸残基的蛋白酶抑制剂如 NEM 或 IAA 等，可以显著抑制 TEV 蛋白酶的酶活性，因为天然的 TEV 蛋白酶含有其维持酶活性所必需的 Cys151。
推荐使用条件
储存缓冲液：25 mmol/L Tris-HCl (pH8.0), 150 mmol/L NaCl, 1 mmol/L EDTA, 5 mmol/L DTT, 50%(v/v) 甘油。
10×TEV Buffer：500 mmol/L Tris-HCl (pH8.0), 250 mmol/L NaCl, 5 mmol/L EDTA, 10 mmol/L DTT。
储存条件
温度：-25℃ ~ -5℃ 保存（注：避免温度低于 - 25℃发生冻结；若长时间冻结会导致活性部分丧失。）
有效期：24 个月。
TEV Protease (His+GST-tag) is a cysteine protease recombinantly expressed in E. coli, carrying dual His and GST tags and derived from Tobacco Etch Virus (TEV). It specifically recognizes the heptapeptide sequence Glu-Asn-Leu-Tyr-Phe-Gln-Gly/Ser and cleaves between the Gln and Gly/Ser amino acid residues. It is widely used to remove GST, His and other affinity tags from fusion proteins. After cleavage of the above heptapeptide sequence by TEV Protease (His+GST-tag), only one extra Gly or Ser residue remains at the N-terminus of the target protein, which minimizes interference with the structure and biological function of the target protein. Another prominent advantage of TEV Protease is its high enzymatic activity in the presence of up to 400 mM imidazole. For most His/GST-tagged target proteins purified via Ni-affinity chromatography, TEV Protease can be directly added to the protein solution containing high-concentration imidazole, and cleavage can be performed simultaneously during dialysis at 4 °C to remove imidazole. Alternatively, cleavage can be carried out post-dialysis for tag removal.
TEV Protease (His+GST-tag) is suitable for cleavage of recombinant proteins fused with His or GST tags. Post-cleavage, uncleaved recombinant protein, excised His tags, excised GST tags, as well as the His+GST-tagged TEV Protease can all bind to His or GST affinity media and be eliminated, with the desired target protein recovered in the flow-through fraction.
Applications
An essential tool protease for tag cleavage in downstream bioengineering workflows.
Suitable for cleavage of fusion proteins containing TEV protease recognition sites.
Recommended Cleavage Protocol
| Component | Volume/Quantity |
|---|
| Fusion Protein | 0.1 mg | | TEV Protease | 20 U | | 10× TEV Buffer | 10 μL | | Deionized Water | Bring total volume to 100 μL | | Incubation Temperature (°C) | 20–37 °C | | Incubation Time (h) | 12–16 |
|
Precautions：
1. The optimal reaction temperature for TEV Protease is 30 °C; it retains high activity between 29 °C and 34 °C. Enzymatic activity drops sharply at temperatures ≥37 °C. To preserve the native structure and bioactivity of target proteins, overnight cleavage at 4 °C is recommended for routine operations. TEV Protease remains active within pH 6.0–9.0, and completely loses activity at pH ≤ 5.
2. TEV Protease activity is not inhibited by common serine protease inhibitors including PMSF, AEBSF, Bestatin, Pepstatin, E-64, TLCK and EDTA. In contrast, cysteine-targeted protease inhibitors such as NEM and IAA strongly suppress TEV protease activity, as native TEV protease contains Cys151, a cysteine residue essential for catalytic function.
Recommended Buffers
Storage Buffer: 25 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1 mmol/L EDTA, 5 mmol/L DTT, 50% (v/v) glycerol
10× TEV Cleavage Buffer: 500 mmol/L Tris-HCl (pH 8.0), 250 mmol/L NaCl, 5 mmol/L EDTA, 10 mmol/L DTT
Storage Conditions
Storage Temperature: -25 °C ~ -5 °C
Note: Avoid freezing at temperatures below -25 °C; prolonged freezing causes partial loss of enzymatic activity.
Shelf Life: 24 months.
ZJ26F0938951
危险品化学品经营许可证（带存储）