– Enterokinase (EK) is a duodenal protease involved in digestion by activating trypsinogen into trypsin, which in turn activates other pancreatic enzymes. EK specifically cleaves after the sequence Asp-Asp-Asp-Asp-Lys (DDDDK↓) and is inactive if a proline follows the site. Recombinant bovine enterokinase (rbEK) shows higher activity than EK from other species and is widely used in biochemical applications. It is a glycosylated single-chain protein of ~200 amino acids, tagged with C-terminal 6×His for Ni²⁺ affinity purification and easy removal after digestion. The purified rbEK has a molecular weight of ~40 kDa and is fully biologically active.