TEV Protease

What it means: Recognition Sequence is the amino acid motif the protease reads before cutting. This enzyme recognises the seven-residue sequence Glu-Asn-Leu-Tyr-Phe-Gln and cleaves the bond after the glutamine, before a glycine or serine.

Why it matters:

• The motif is rare in natural proteins, so a fusion construct can be cut at exactly one place without damaging the target.
• Cleavage occurs between Gln and Gly/Ser, so the released protein retains only that single residue from the site — close to a native N-terminus.
• The site must be engineered into the construct deliberately; it will not act on an untagged protein.

How to interpret here: Place the ENLYFQ↓G/S site between the affinity tag and the target when designing the construct. Either the Gly or the Ser variant works.

Tip: If cleavage is incomplete, check that the site is accessible — a buried linker cuts far more slowly than one in a flexible region.

What it means: Enzyme Variant describes how this preparation differs from the native protease. abm describes it as an improved version of the Tobacco Etch Virus protease, with enhanced activity, stability and site-specificity relative to the native form.

Why it matters:

• Higher activity means a smaller enzyme-to-substrate ratio reaches the same cleavage, which leaves less protease to remove afterwards.
• Better stability supports the long, cold digests that fragile target proteins need, without the enzyme falling off over the incubation.
• Improved site-specificity is what keeps cleavage confined to the engineered site rather than nicking the target elsewhere.

How to interpret here: Protocols written for native TEV protease can usually be shortened or run at lower enzyme loading. Under optimal conditions up to 99% cleavage is achieved in 1–2 hours.

Tip: For difficult substrates, raise the enzyme-to-substrate ratio before extending the incubation — this variant tolerates it.

What it means: Operating Temp is the range over which the protease stays active. It works from 4°C to 30°C, with an optimum at 30°C, and across pH 5.5–9.0.

Why it matters:

• A wide range means the digest can be matched to the stability of the target protein rather than the other way round.
• Fragile or aggregation-prone targets can be cleaved overnight at 4°C instead of being held at room temperature.
• Broad pH tolerance means the digest can often be done in the elution buffer from the preceding purification step, without a buffer exchange.

How to interpret here: Use 30°C for speed when the target tolerates it — up to 99% cleavage in 1–2 hours. Drop to 4°C and extend the time for unstable proteins.

Tip: Check that the buffer sits inside pH 5.5–9.0 before adding enzyme; outside it the digest simply will not proceed.

What it means: Cofactors Included lists what ships alongside the enzyme — 1.0 ml of 20X TEV Protease Reaction Buffer and 500 µl of 100 mM DTT.

Why it matters:

• TEV protease is a cysteine protease: its active-site thiol must stay reduced, so DTT is required rather than optional.
• Supplying DTT separately from the buffer lets it be added fresh, since reducing agents oxidise in storage and lose potency.
• The 20X buffer is concentrated, so it contributes little volume to the digest — useful when the protein sample is already dilute.

How to interpret here: Add the DTT fresh at each digest rather than premixing it into the buffer for long-term storage. Note that carried-over DTT may interfere with downstream steps such as nickel affinity capture.

Tip: If the target protein has disulfide bonds, the required DTT will reduce them too — plan the cleavage step accordingly.