Expression and purification of soluble and active human enterokinase light chain in .

Human enterokinase light chain (hEK) specifically cleaves the sequence (Asp)-Lys↓X (DK), making this a frequently used enzyme for site-specific cleavage of recombinant fusion proteins. However, hEK production from is limited due to intramolecular disulphide bonds. Here, we present strategies to obtain soluble and active hEK from by expressing the hEK variant C112S fused with maltose-binding protein (MBP) through DK and molecular chaperons including GroEL/ES. The fusion protein self-cleaved thereby removing the MBP in the cells. Thus, the self-cleaved hEK variant was released into the culture medium. One-step purification using HisTrap™ chromatography purified the hEK variant exhibiting an enzymatic activity of 3.1 × 10 U/mL (9.934 × 10 U/mg). The approaches presented here greatly simplify the purification of hEK from without requiring refolding processes.