Ezcut TEV Protease Recombinant Protein

Liquid
1mg/ml solution in 0.1 M Tris-HCl, 0.5 M NaCl, 20% glycerol, 5 mM DTT and 0.5 mM EDTA, pH 8.0

>=95% by SDS-PAGE

1 mg/ml (lot specific)

The EZCut TEV Protease has an activity of >=10,000 units/mg.

Store at -80°C. Stable for at least 1 year as supplied. It may be further diluted to 0.1-0.5 mg/ml with 0.1M Tris-HCl, 0.5M NaCl, 20% glycerol, and stored at -80°C in aliquots. Avoid repeated freezing and thawing cycles.

28.6 kDa (2038-2279 aa + C-terminal poly-his tag).

One unit is defined as the amount of EZCut TEV Protease required to cleave >90% of 10 ug a control protein substrate (containing an ENLYFQS sequence at N-terminus) in 1 h at 37°C.

E.coli

In order to find the optimum cleavage conditions for a target fusion protein, it is recommended to run preliminary cleavage reactions at a small scale. Successful cleavage with TEV protease is dependent upon proper folding of the fusion protein that enables access of the TEV recognition sequence by the enzyme. The target fusion protein (1 mg/ml) containing TEV protease cleavage site should be purified to homogeneity. Recommended cleavage buffer is 50 mM Tris, buffer, 0.1 M NaCl, pH 8.0.
1.To 10 ul of target protein in an Eppendorf tube add 0.05-0.5 ug (0.5-5 U) of TEV protease.
2.Add 10 ul of freshly prepared 50 mM Tris, buffer, 0.1 M NaCl, pH 8.0 containing 5 mM DTT. Mix well by pipetting up and down; incubate at 37°C for 1 h. Analyze by SDS-PAGE.
3.Once optimum cleavage conditions are obtained, the reaction can be scaled up to cleave the entire amount of the target fusion protein. After completion of cleavage reaction, TEV can be removed by passing the entire mixture through Ni-chelating resin. Collect the flow through and wash with the resin with 50 mM Tris, buffer, 0.1 M NaCl, pH 8.0. Combine the washes with the flow through to obtain cleaved protein product.
4. Optimum temperature for TEV protease cleavage is 34°C. However, the reaction can also be carried out at 37°C or 30°C also. If the target protein is not stable at these temperatures, the cleavage reaction can also be performed at room temperature or 4°C. In such cases, the amount of TEV protease should be optimized for different target proteins

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