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 (D(4)K), 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 D(4)K 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 × 10(3) U/mL (9.934 × 10(5) U/mg). The approaches presented here greatly simplify the purification of hEK(L) from E. coli without requiring refolding processes.