Tobacco Etch Virus Proteinase, mutant (mTEVP, Recombinant) is used as a valuable tool for cleaving affinity tags and fusion proteins in recombinant protein purification protocols. The wild type TEVP contains a self-cleavage site that the protein recognizes and cleaves. Since the protein cleaves itself, it is inherently unstable. A mutant TEVP has been generated and tested to have reduced self-cleavage function with no reduction in its target cleavage activity. The protein was expressed in bacteria and purified by Ni affinity chromatography.
Application:
Suitable for use in cleaving 6X His-tags from target proteins. After digestion, the cleaved tag and the mTEVP, since they both have His-tags, can be removed by adsorption onto a Ni column. Can also be used to remove Protein A tags.
Recognition Sequence:
E-N-L-Y-F-Q*G-M------
*-cleavage point
Purity:
Purified by nickel chromotography ((same/more than) 90%). Single band (SDS-PAGE) of ~28kD
Activity:
mTEVP has a cleavage activity of ~8 units/ul
Unit Definition:
One unit of mTEVP cleaves (same/more than) 95% of 3ug of control substrate in 1hour at 30 degrees C.
Recommended reaction buffer:
TEV enzyme assay is carried out in 50mM Tris buffer, pH 8.0, 0.5mM EDTA, 1mM DTT.
Optimal Reaction Temp:
30 degrees C (minimal temp is not tested)
reaction buffer:
50 mM Tris buffer, pH 8.0, 0.5 mM EDTA and 1 mM DTT.
Storage Buffer:
100mM Tris, pH 8.5, 500mM sodium chloride, 50% glycerol, 5mm DTT, 0.5mM EDTA
Storage and Stability:
May be stored at 4 degrees C for at least 6 months. For long-term storage, aliquot and store at -70 degrees C. Aliquots are stable for 12 months at -70 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
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