Helpful tips

Does TEV protease have a His tag?

Does TEV protease have a His tag?

TEV Protease is a restriction grade protease that has robust activity at 4°C with high specificity and great stability. The optimal temperature for cleavage with this enzyme is 34°C. It contains a C-terminal His tag and can be easily removed after cleavage reactions by passing the reaction through a Ni-chelating resin.

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.

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 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). Due to its high sequence specificity it is frequently used for the controlled cleavage of fusion proteins in vitro and in vivo.

How do I get rid of his tag?

The easiest way is to use TEV protease (very specific) which also has a HIS tag itself. After cleavage (30C 4h or 4C overnight) you can run another Ni-NTA column, but this time, collect the flow-through, because the column will bind the cleaved tag and TEV protease itself. For us it works perfectly fine.

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).

Is protease a protein?

Proteases, being themselves proteins, are cleaved by other protease molecules, sometimes of the same variety. This acts as a method of regulation of protease activity. Some proteases are less active after autolysis (e.g. TEV protease) whilst others are more active (e.g. trypsinogen).

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.

Where does TEV protease cleave?

It is also relatively easy to overproduce and purify large quantities of the enzyme. 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 Flag tag sequence?

The Flag® tag, also known as the DYKDDDDK-tag, is a popular protein tag that is commonly used in affinity chromatography and protein research for over 20 years now (6,7,8,9,10,11). As its second name suggests the tag consists of an amino acid sequence DYKDDDDK. (D=Aspartic acid; K=Lysine; Y=Tyrosine).

How do you remove a protein tag?

The tag may be removed by cleavage of a protease site placed between the target protein and the affinity tag, followed by a step to separate the protein and affinity tag.