What is TEV protease cleavage site?
The cleavage site The TEV protease prefers for recognition ENLYFQ|S(G,A) sequence, but it was shown that it can also cleave other motives with substitutions in the positions P2, P4, and P5 (EP5P4YP2Q|S(G,A)) which to some extent correspond with cleaving variability of natural enzyme in these sites.
Where does TEV cleave?
Recombinant Tobacco Etch Virus (TEV). The optimum recognition site for this enzyme is the sequence 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.
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.
What is a TEV site used for?
It is often used for the removal of affinity purification tags such as maltose-binding protein (MBP) or poly-histidine from fusion proteins. TEV Protease has a 7xHis-tag for easy removal from a reaction using nickel affinity resins and has been engineered to improve thermal stability and decrease autolysis.
Is TEV a cysteine protease?
Tobacco etch virus (TEV) protease is a cysteine protease exhibiting stringent sequence specificity. The enzyme is widely used in biotechnology for the removal of the affinity tags from recombinant fusion proteins.
Does TEV protease have a His tag?
It contains a C-terminal His tag and can be easily removed after cleavage reactions by passing the reaction through a Ni-chelating resin.
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 store TEV protease?
We store TEV at -20 C. It doesn’t freeze, so I can take out what I need whenever I have to cleave my fusion protein. Having a poly-Arg tail on your TEV construct helps keeping it soluble as well.
What is TEV sequence?
TEV protease (EC 3.4. 22.44, Tobacco Etch Virus nuclear-inclusion-a endopeptidase) is a highly sequence-specific cysteine protease from Tobacco Etch Virus (TEV). It is a member of the PA clan of chymotrypsin-like proteases.
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).
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.