Тев протеаза от табака вирус Etch (TEV) используемый для расщепления сплавленных белков и удаления бирок из рекомбинантного

Описание и отзывы

Характеристики

TEV protease is cysteine protease from Tobacco Etch Virus (TEV) which is highly used for the cleavage of fusion proteins and removal of tags from recombinant proteins in vitro or in vivo. This enzyme belongs to chymotrypsin-like proteases and shows high sequence specificity.

TEV protease recognition sequence with the highest catalytic efficiency is ENLYFQ ↓(G/S).The cleavage site is between glutamine and glycine/serine. Seven-amino acid sequence commonly used is Glu- AsN-LEu -Tyr- PHE-GLN ↓-Gly. 

TEV protease reaches its best activity at pH 7.0 and 30℃.However,it also is active in a wide range of buffers of pH 6.0 to 8.5 and temperature 4 to 30℃. So, it is flexible to select the reaction conditions based on the target protein. In addition,TEV Protease contains a 6x His-Tag on the n-terminal for easy removal by Ni-NTA resin to purify the target protein.

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Information

Storage 

Transportation with ice pack.

Stored at -20℃, stable for 6 months; Stored at-70℃,stable for one year.  It is recommended to subpackage to avoid repeated freeze-thaw. 

Enzyme activity

1 unit refers to shear more than 85% of 2 μg substrate required enzyme amount in 1× TEV Buffer(50 mM Tris, pH 8.0, 0.5 mM EDTA, 1 mM DTT), reaction at 30℃ for 1h.

Application method

1.Reaction system in EP tube: 

2. Incubate at 30℃, sucking out 30 μL of the reaction solution at 1, 2, 4 and 6 h, put respectively into separate EP tubes.

3. Add 30 μL 2×SDS Loading Buffer to the EP tube and place it at -20℃.

4. After reacted, all the samples were boiled for 5 min, then 40 μL was taken out  for SDS-PAGE analysis. Determine the best reaction time. If the fusion protein requires low temperature treatment, the reaction solution can be placed at 4℃ to prolong the reaction time and increase the amount of TEV enzyme to ensure the digestion effect.

Notes

Factors affecting natural TEV enzyme activity: 

1) PMSF and AEBSF (1 mM), TLCK (1 mM), Bestatin (1 mg/mL), EDTA (1 mM), Pestatin A (1 mM) and E-64 (3 mg/mL)

2) Zinc ion concentration (> 5 mM); 

3) Reagents that react with cysteine are potential inhibitors of TEV.

FOR RESEARCH USE ONLY

References

1.Tobacco etch virus (TEV) protease with multiple mutations to improve solubility and reduce self cleavage exhibits enhanced enzymatic activity . Heejin Nam, Beom J. wang,  Deog Young Choi,  Sooim Shin,et al.  FEBS Open Bio. 2020 Apr; 10(4): 619-626.

2. Differential temperature dependence of tobacco etch virus and rhinovirus 3C protease. Raran-Kurussi, S., Tözsér, J., Cherry, S., Tropea, J. E. & Waugh, D. S. s. Anal. Biochem. 436, 142-144 (2013).

3.  TEV protease facilitated stoichiometric delivery of multiple genes using a single expression vector. Chen X, Pham E and Truong K. Protein Sci 19, 2379-2388. 

4. Improved solubility of TEV protease by directed evolution. Van den Berg S, Lofdahl PA, Hard T and Berglund H (2006)  J Biotechnol 121, 291-298.