Earticle

초록

영어

Solid-phase refolding of immobilized proteins can be an effective way to reuse an immobilized enzyme column. Oriented immobilization methods are known to provide higher activity of the immobilized enzymes. In this study, using recombinant EK (enterokinase) as a model enzyme and 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, as a model substrate, we evaluated two oriented immobilization methods, i. e., reductive alkylation of N-terminus α-amine and affinity interaction between poly-histidine tag and Ni-NTA (nickel-nitrilotriacetic acid). The immobilization yield, activity and cleavage of the immobilized enzymes, and the yield
of solid-phase refolding were compared. The Ni affinity immobilization and the covalent immobilization yields were about 100% and 65%, respectively. But the specific activities were the same, about 50% of that of the soluble enzyme. The cleavage rate by the covalently immobilized EK was higher than the soluble enzyme and the side reaction of cryptic cleavage was significantly decreased. Covalently immobilized EK showed almost 100% refolding yield but the affinity immobilized EK showed only 70% yield, which suggested the covalent conjugation provided more rigid ‘reference structure' for the solid-phase refolding. The monomeric hGH could be easily obtained by capturing the cleaved poly Histidine tag by the Ni affinity column.

한국어

융합단백질의 절단을 위해 EK를 고정화하여 액상 절단반응과 같은 80%의 절단수율을 얻을 수 있었다. 그리고 니켈친화칼럼을 이용하여 간단한 정제공정을 구축하였다. 공유결합한 EK의 경우 니켈친화 결합한 EK보다 높은 재접힘 수율을 나타내었고 풀림과 재접힘을 이용하여 효소의 초기 활성을 회복함에 따라서 반복사용을 통한 경제적인 절단공정을 구축할 수 있게 되었다. 그러나 고정화 과정에서 효소의 활
성이 감소하는 문제점과 고정화 수율을 높이기 위한 연구가 필요하다.

목차

Abstract
 서론
 재료 및 방법
  실험재료
  효소 고정화 방법
  Enterokinase의 활성측정
  융합단백질 절단반응
  목적단백질의 분리정제
  고정화 효소의 풀림과 재접힘
 결과 및 고찰
  EK 고정화
  융합단백질의 절단반응
  고정화 EK의 풀림과 재접힘
  목적단백질의 정제
 요약
 REFERENCES

키워드

저자

• 서창우 [ Chang-Woo Suh | 한양대학교 화학공학과 생물공정연구실 ]
• 나세진 [ Sea-Jin Na | 한양대학교 화학공학과 생물공정연구실 ]
• 박신혜 [ Sin-Hye Park | (주)대웅 생명공학연구소 ]
• 박승국 [ Seung-Guk Park | (주)대웅 생명공학연구소 ]
• 이은규 [ Eun-Kyu Lee | 한양대학교 화학공학과 생물공정연구실 ] Corresponding author

참고문헌

발행기관

간행물

표지보기 관심저널 등록

• 간행물명

  KSBB Journal

• 간기

  격월간

• pISSN

  1225-7117

• 수록기간

  2009~2013

• 십진분류

  KDC 476 DDC 576