What does TEV protease do?

What does TEV protease do?

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 cleave?

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 the TEV site?

Origin. The tobacco etch virus encodes its entire genome as a single massive polyprotein (350 kDa). This is cleaved into functional units by the three proteases: P1 protease (1 cleavage site), helper-component protease (1 cleavage site) and TEV protease (7 cleavage sites).

How much TEV protease should I use?

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 does TEV protease cleave?

TEV protease specifically cleaves proteins within a seven-residue optimal recognition. sequence. This sequence is: Glu-Asn-Leu-Tyr-Phe-Glu-Gly-Ser (ENLYFQ↓G/S) The 7th residue can be either Gly (G) or Ser (S), and proteolytic cleavage occurs.

What is the amino acid sequence of TEV protease?

ENLYFQG/S
Tobacco etch virus (TEV) protease is a 27‐kDa catalytic domain of the polyprotein nuclear inclusion a (NIa) in TEV, which recognizes the specific amino acid sequence ENLYFQG/S and cleaves between Q and G/S.

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.

What type of protease is TEV protease?

cysteine protease
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 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.

How do you stop TEV protease?

TEV Protease

1. Removal of affinity purification tags such as maltose-binding protein (MBP) or poly-histidine from fusion proteins.
2. Optimal activity and stability for up to 24 months.
3. Active in a wide range of buffers; optimal activity between pH 6.0 and 9.0.
4. High substrate specificity with no non-specific proteolysis.

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).