Covalent immobilization and solid-phase refolding of enterokinase for fusion protein cleavage

Using recombinant enterokinase (EK) as the model proteolytic enzyme, α-amine-coupling covalent immobilization to glyoxyl agarose was evalulated in terms of immobilization yield and the activity and thus the cleavage performance of the immobilized enzyme. Nearly all the enzyme was immobilized by the covalent conjugation, but the specific activity was only 20-30% of that of the soluble enzyme at various pH conditions. However, the cleavage rate by the covalently immobilized EK was higher than that of the soluble enzyme and the undesirable side reaction, i.e., the cryptic cleavage was significantly reduced. In order to reuse the immobilized EK repeatedly, solid-phase refolding of the immobilized EK was attempted. The covalently immobilized EK showed almost 100% refolding yield whereas the soluble EK showed only ca. 36% yield. It was confirmed that the covalent conjugation maintained the rigid 'reference structure' during a denaturant-induced unfolding step, which would in turn provide for a more efficient route to refolding in the subsequent renaturation step.