New England Biolabs, P8112S, TEV Protease

The TEV protease recognition sequence with the highest catalytic efficiency is ENLYFQ ▼S; however, the amino acid in the P1’ position can also be G, A, M, C, or H (1). Related Categories NEBExpress MBP Fusion and Purification System,, Bacterial E. coli Protein Expression,, Nickel Purification (His-tag), Applications Fusion Protein Cleavage,, Target Protein Insolubility ,, Protein Purification, Specification Unit Definition 1 unit of TEV Protease will cleave 2 µg of MBP-fusion protein, MBP5-TEV-paramyosin ΔSal, to 95% completion in a total reaction volume of 10 μl in 1 hour at 30°C in 50 mM Tris-HCl (pH 7.5 @ 25°C) with 0.5 mM EDTA and 1 mM DTT. Reaction Conditions 1X TEV Protease Reaction Buffer Incubate at 30°C 1X TEV Protease Reaction Buffer 50 mM Tris-HCl 0.5 mM EDTA 1 mM DTT (pH 7.5 @ 25°C) Storage Buffer 50 mM Tris-HCl 250 mM NaCl 1 mM TCEP 1 mM EDTA 50% Glycerol pH 7.5 @ 25°C Heat Inactivation 65°C for 10 minutes Molecular Weight Apparent: 28 kDa Unit Assay Conditions Two fold dilutions of TEV Protease are incubated with 2 μg MBP5-TEV-paramyosin ΔSal and 1X TEV Protease Reaction Buffer in a 10 μl reaction. The reaction mix is incubated at 30°C for 1 hour. Separation of reaction products are visualized by SDS-PAGE. FAQ Q: Can TEV Protease recognize and cleave a sequence other than Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser)? A: The TEV Protease recognition sequence with the highest catalytic efficiency is ENLYFQ▼S. However, the amino acid in the P1’ position can also be G, A, M, C, or H (1). Kapust, R.B. et al. (2002) Biochem. and Biophysical Research Comm. 294, 949-955. Q: Is it necessary to dialyze the sample prior to cleavage with TEV Protease? A: If the fusion protein sample contains >2 M urea, >0.5 M guanidine hydrochloride, >50 mM imidazole, pH values below 6 or above 9, or cysteine protease inhibitors, then it will be necessary to dialyze the fusion protein before TEV Protease cleavage. Q: Can TEV Protease be used at lower temperatures? A: Yes, TEV protease can be used at temperatures below 30ºC. Cleavage at lower temperatures, such as 4ºC, requires overnight incubation. Q: Is TEV Protease compatible with protease inhibitors? A: TEV Protease activity is unaffected by the following protease inhibitors: aprotinin, benzamidine, leupeptin, pepstatin, and PMSF. Q: Is TEV Protease compatible with different reaction buffers? A: An optimal reaction buffer is the supplied 10X TEV Protease Reaction Buffer (0.5M Tris-HCl pH 7.5, 5mM EDTA, 10mM DTT). However, the enzyme is also active in HEPES, potassium phosphate and sodium acetate buffers between pH values of 6 and 9. Q: Can TEV Protease be removed from the reaction after cleavage? A: Yes, TEV Protease contains a polyhistidine tag at its N-terminus. After cleavage of the fusion protein, remove TEV Protease from the cleavage reaction by immobilized metal affinity chromatography.