Expression and purification of soluble and active human enterokinase light chain in Escherichia coli

Human enterokinase light chain (hEK_L) specifically cleaves the sequence (Asp)₄-Lys↓X (D₄K), making this a frequently used enzyme for site-specific cleavage of recombinant fusion proteins. However, hEK_L production from Escherichia coli is limited due to intramolecular disulphide bonds. Here, we present strategies to obtain soluble and active hEK_L from E. coli by expressing the hEK_L variant C112S fused with maltose-binding protein (MBP) through D₄K and molecular chaperons including GroEL/ES. The fusion protein self-cleaved in vivo, thereby removing the MBP in the E. coli cells. Thus, the self-cleaved hEK_L variant was released into the culture medium. One-step purification using HisTrap™ chromatography purified the hEK_L variant exhibiting an enzymatic activity of 3.1 × 10³ U/mL (9.934 × 10⁵ U/mg). The approaches presented here greatly simplify the purification of hEK_L from E. coli without requiring refolding processes.