융합단백질 절단반응을 위한 고정화된 enterokinase의 고체상 재접힘

Abstract

We immobilized recombinant enterokinase (EK) by covalent attachment to glyoxyl Sepharose through reductive alkylation of N-teminus ε-amine and by affinity interaction between poly His tagged EK to Ni-NTA resin. And We evaluated its performance to cleave a fusion protein, that consisted of recombinant human growth hormone and six His tag that was linked by the peptide of EK-specific recognition sequence. The Ni affinity immobilized yield was nearly 100% but the specific activity was 50%. And covalent immobilized yield was 65% and specific activity was remained only 49%. The cleavage rate by the covalently immobilized EK was higher than the soluble enzyme and the side reaction of cryptic cleavage was significantly decreased. We compared solid-phase refolding yield of affinity immobilized EK with covalent immobilized EK. Covalent immobilized EK showed almost 100% refolding yield but affinity immobilized EK showed only 70% refolding yield. The monomeric hGH could be easily obtained by capturing the cleared poly His tag by Ni affinity column.