Enterokinase Cleavage Enzyme (Mammalian Produced)

What it means: Recognition Sequence is the motif the enzyme reads before cutting. Enterokinase recognises Asp-Asp-Asp-Asp-Lys (DDDDK) and cleaves immediately after the C-terminal lysine.

Why it matters:

• Cutting after the lysine means the released protein inherits no residues from the recognition sequence — a 100% native N-terminus, unlike proteases that cut inside their site.
• DDDDK is part of the FLAG octapeptide (DYKDDDDK), so the same tag can serve for antibody detection, Western blot and anti-FLAG purification before being removed.
• The motif must be engineered between the tag and the target; the enzyme will not act on an untagged construct.

How to interpret here: Place DDDDK directly before the first residue of the desired product. Whatever follows the lysine becomes the new N-terminus exactly as designed.

Tip: If cleavage is slow, check the site is in a flexible, solvent-exposed linker — a buried DDDDK cuts poorly regardless of enzyme amount.

What it means: Expression System is where the enzyme itself was produced. This enterokinase is made in a human cell line grown in serum-free media, rather than in bacteria or yeast.

Why it matters:

• Mammalian production gives the enzyme its appropriate glycosylation, which is what supports accurate cleavage with minimal non-specific activity — even at 20x concentration.
• Serum-free culture means the preparation is endotoxin free, which matters when the cleaved product is destined for cell-based assays or animal work.
• Bacterially produced enterokinases more often carry endotoxin and show higher off-target cleavage.

How to interpret here: Choose this enzyme when the cleaved protein must stay endotoxin free, or when a previous digest damaged the target through non-specific cutting.

Tip: Record the mammalian, serum-free origin in your methods where endotoxin status has to be documented.

What it means: Shelf Life is the period over which the manufacturer guarantees stated performance — here 3 years from the shipping date when stored and handled as instructed at −20°C.

Why it matters:

• Three years is unusually long for an enzyme in this range, so a single lot can cover a multi-year purification programme.
• The clock starts at shipping, not first use, so a vial in a shared freezer may have less usable life than it appears to.
• The guarantee is conditional on aliquoting: repeated freeze-thaw shortens it regardless of the date.

How to interpret here: Briefly centrifuge the vial before first opening, split the stock into working aliquots, and label them with the receipt date so the remaining window stays visible.

Tip: Aliquot at a volume matched to a single digest — partial thaws are what actually consume the shelf life.

What it means: Operating Temp gives the reaction conditions the supplied buffer is built around — 25°C at pH 8.0. Separately, the enzyme remains active over a broad range and completes the reaction anywhere from 4°C to 60°C.

Why it matters:

• The recommended condition is where cleavage efficiency and specificity were characterised, so it is the right starting point.
• The wide tolerance means the digest can be matched to the stability of the target rather than forcing the target to tolerate the enzyme.
• Fragile or aggregation-prone proteins can be cleaved slowly in the cold; robust ones can be pushed warmer to save time.

How to interpret here: Start at 25°C, pH 8.0 in the supplied 10X buffer. Move colder for unstable targets and accept a longer incubation; 95% cleavage in 1 hour is achievable under recommended conditions.

Tip: Change one variable at a time — temperature or enzyme ratio, not both — or an improved digest will not tell you why.