EK initiates activation of pancreatic proteases by converting trypsinogen to trypsin, which in turn activates chymotrypsin, carboxypeptidases and elastases. Located in intestinal brush border, it is a disulfide bond linked dimer of the heavy and light chains, which are derived from the same single-chain precursor. The multidomain‑containing heavy chain consists of a short cytoplasmic tail, a transmembrane, a SEA, a SRCR, a MAM, two CUB and two LDL-receptor class A domains. The light chain contains the catalytic domain of trypsin-like serine proteases. The purified recombinant bovine EK (residues 788-1035) corresponds to a disulfide bond‑linked dimer that consists of the C-terminal fragment of the heavy chain (residues 788-800) and the light chain (residues 801-1035). rbEnterokinase can cleave fusion proteins having an accessible Enterokinase cleavage site (DDDDK). At an average ratio for fusion protein:rbEnterokinase of 1000:1 (w/w), cleavage up to 90% completion is achieved within one hour at room temperature. Non-specific cleavage at basic residues has also been observed for some proteins. It is recommended that cleavage reaction be optimized for each fusion protein. The reaction may be terminated by passing the sample through a soybean trypsin inhibitor (SBTI)-agarose affinity column (e.g. Sigma Catalog # T0637 ) to remove the rbEnterokinase from the reaction mixture. SBTI inhibits rbEnterokinase with a K_i of 1.6 nM.