The tobacco etch virus (TEV) protease is a useful tool for the removal of fusion tags from recombinant proteins. TEV protease has a strict 7 amino acid cleavage recognition sequence of Glu-Asn-Leu-Tyr-Phe-Gln-Gly/Ser [ENLYFQ(G/S)] and cleavage occurs between the Gln and Gly/Ser residues, The most commonly used sequence is ENLYFQG. It is recommended that the cleavage for each fusion protein be optimized by varying the amount of recombinant viral TEV protease, reaction time, or incubation temperature. It can be removed by Ni2+ affinity resin. Recombinant Tobacco Etch Virus Protease (rTEV) has (NIa) protease catalytic domain which corresponds to a molecular weight of 28 kDa. It is unique with high specificity and is active at low temperature. rTEV Protease has a 6*His-tag for easy removal from a reaction using nickel affinity resins and has been engineered to improve thermal stability and decrease autolysis.
1. Add the following to a microcentrifuge tube:
Fusion Protein | 15μg
|
10X rTEV Protease Buffer +/– Salt
| 5μl |
rTEV Protease
|
5U
|
ddH2O
|
To 50μl
|
2. Mix and incubate at 30°C, Remove 10 μL aliquots
at 1, 2, 4, and 6 hours.
3. Analyze by SDS-PAGE.
Guidelines for Cleavage
- For most fusion proteins, TEV Protease functions
optimally in a reaction mixture without NaCl; however, conditions may be
optimized by varying the NaCl concentration from 0 mM to 500 mM. Remember to
take into account the contribution of salt from the enzyme and from your
substrate. When setting up your cleavage reaction, use the appropriate 10X rTEV
Protease Buffer +/- Salt.
- Researchers
need to optimize their specific reaction conditions. As an initial suggestion, 10
units of rTEV protease can be used per 30μg of target protein for 1 hour at 30
°C, or overnight at 2–8 °C. The cleavage efficiency can then be estimated by
SDS-PAGE.
One unit of TEV protease cleaves ≥85% of 3 μg of control substrate in 1 hour at pH 8.0
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