EvoBio Recombinant Enterokinase – 500 Units

Product Overview

Enterokinase is a highly specific protease that cleaves peptide bonds immediately after a lysine that is present after four aspartic acids as shown Asp-Asp-Asp-Asp-Lys¯. The enzyme does not function if this recognition site is followed by a proline residue, and in certain cases may cleave other basic amino acids depending on the substrate’s conformation. EvoBio Recombinant Enterokinase is a bovine protein containing an active light chain engineered as a single polypeptide, retaining full biological activity. It has a molecular mass of 27.6 kDa and is produced by expressing the cloned bovine enterokinase light-chain gene in E. coli, followed by purification using EvoBioSystem’s proprietary chromatographic methods. Molecular Weight: 27.6 kDa Storage: -20ºC. Mix and centrifuge the tube before use. Applications: Removal of affinity purification tags containing an enterokinase cleavage site. Storage Buffer Composition: The enzyme is provided in a storage buffer containing 20 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl₂, and 50% glycerol, adjusted to pH 8.0 at 25°C.

Quality Control Assays Protein concentration is quantified by measuring absorbance at 280 nm. A 12% Tris-Glycine SDS-PAGE analysis verifies a purity level of ≥ 95%. The enzyme exhibits a specific activity of ≥ 10 U/µL protein. One unit of enzyme is defined as the amount of enzyme required to achieve 95% cleavage of 25 µg MBP- EK-paramyosin-ΔSal substrate within 16 hours at 25°C in a total reaction volume of 25 µL.

Cleavage Optimization Guidelines To determine the best conditions for cleaving a target fusion protein, it is advisable to begin with small- scale test reactions. Efficient enterokinase activity requires the fusion protein to be correctly folded so the enzyme can access its recognition site. For optimal results, the fusion protein containing the enterokinase cleavage sequence should be purified to homogeneity.

Cleavage Procedure 1. Prepare the Reaction Buffer: • 20 mM Tris-HCl (pH 8.0) • 50 mM NaCl • 2 mM CaCl₂

2. Add 25 µg of the sample to the reaction buffer. 3. Adjust the final volume to 20 µL using nuclease-free water. 4. Add 1 µL of EvoBio Recombinant Enterokinase Light Chain. 5. Incubate the reaction at 25°C for 16 hours.

Note • Enterokinase activity decreases at high salt levels; NaCl should be kept at 100 mM or below. • The reaction buffer should maintain a pH between 6 and 9. • 2 mM Ca²⁺ is required for optimal enzymatic activity.

Enzyme Removal Options • Enterokinase can be selectively captured using trypsin inhibitor agarose or benzamidine sepharose. • EvoBio Recombinant Enterokinase carries a C-terminal His-tag, allowing efficient removal through standard nickel affinity chromatography.

For research use only, not for clinical diagnosis.