Solid-phase refolding of immobilized enterokinase for fusion protein cleavage

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. EK를 고정화하기 위해 니켈 친화결합 방법과 공유 결합형 고정화 방법을 수행하였으며 니켈 친화결합이 공유 결합형 고정화보다 높은 고정화 수율과 activity를 나타냈다. 풀림과 재접힘을 이용한 효소의 활성 회복은 공유결합형 고정화가 니켈 친화결합보다 높은 결과를 나타내었다. 또한 기질의 분자량 크기에 따른 절단율의 차이가 없었으므로 레진 공극 내부로의 확산도 차이에 의한 절단반응의 차이는 없는 것으로 나타났고, 기질 종류에 따른 EK의 활성은 작은 기질이 큰 기질보다 높은 활성을 보였다.