What does TEV protease cleave?
The 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.
Where does TEV protease cleave?
What is the cleavage site for TEV protease? TEV protease recognizes a linear epitope of the general form E-Xaa-Xaa-Y -Xaa-Q-(G/S), with cleavage occurring between Q and G or Q and S. The most commonly used sequence is ENLYFQG.
How do you use TEV protease?
How Much TEV Protease to Use? Rule of thumb: Use 1 µg TEV protease per 25 µg to 100 µg of substrate (minimum enzyme concentration: 1 unit/mL). Use more enzyme, if the cleavage site of the substrate is occluded sterically or the substrate is aggregated.
What is TEV recognition site?
TEV Protease is a highly specific cysteine 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).
How do you make TEV Protease?
Combine 15 µg fusion protein and H2O to make a total reaction volume of 45 μl. Add 5 µl TEV Protease Reaction Buffer (10X) to make a 50 μl total reaction volume. Add 1 µl TEV Protease . In a separate tube, combine 5 µg fusion protein , 5 µl TEV Protease Reaction Buffer (10X) and H2O to a volume of 50 μl.
Does TEV Protease have a His tag?
BACKGROUND: BioVision’s EZCut™ TEV Protease is a cysteine protease that recognizes the cleavage site of Glu-Xaa- Xaa-Y- Xaa- Gln-(Gly/Ser) and cleaves between Gln and Gly/Ser. It contains a C-terminal His tag and can be easily removed after cleavage reactions by passing the reaction through a Ni-chelating resin.
How do you purify TEV Protease?
The His-tagged TEV protease can be purified in two steps using immobilized metal affinity chromatography (IMAC) followed by gel filtration.
How do you get rid of TEV Protease?
TEV Protease contains a polyhistidine tag at its N-terminus and can be removed from the reaction by immobilized metal affinity chromatography, such as NEBExpress Ni-NTA Magnetic Beads (NEB #S1423), NEBExpress Ni Spin Columns (NEB #S1427), or NEBExpress Ni Resin (NEB #S1428).
Does Pmsf inhibit TEV protease?
TEV protease activity is not inhibited by PMSF and AEBSF (1 mM), TLCK (1 mM), Bestatin (1 mg/mL), pepstatin A (1 mM), EDTA (1 mM), E-64 (3 mg/mL), or “complete” protease inhibitor cocktail (Roche).
How do you get rid of TEV?
How do you stop TEV protease?
- Removal of affinity purification tags such as maltose-binding protein (MBP) or poly-histidine from fusion proteins.
- Optimal activity and stability for up to 24 months.
- Active in a wide range of buffers; optimal activity between pH 6.0 and 9.0.
- High substrate specificity with no non-specific proteolysis.
How do you inhibit TEV protease?
TEV protease is inhibited by reaction buffers containing >40% Glycerol. Inhibition occurs in the presence of ≥ 5 mM Zn2+, ≥ 1 mM Cu2+ and ≥ 10 mM Co2+. Compatible with 10mM MgSO4, MnCl2 and CaCl2 and up to 100mM EDTA.
What is TEV protease recombinant?
Description: TEV Protease, Recombinant (rTEV) is a site-specific protease purified from. E. coli by the affinity tag, polyhistidine tag. The protease can be used for the removal of affinity tags from fusion proteins.
What is the history of TEV protease plasmids?
Plasmids containing different TEV protease versions were episomally introduced in a pRSII413 vector which was a gift from S. Haase (Addgene no. 35450). Transformed cells containing the HIS3 gene were selected on SMM plates (SMM with 20 g l –1 agar) and propagated in SMM at 30 °C.
How much MBP does TEV protease cleave?
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.
How do you prepare TEV protease reaction mix?
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.