화학공학소재연구정보센터
홈 로그인 로그아웃 연락처 사이트맵
Journal of the Korean Industrial and Engineering Chemistry, Vol.14, No.4, 458-463, June, 2003
Export Citation
융합 단백질을 이용한 B30-homoserine 인슐린 전구체의 발현과 분리·정제
Expression and Purification of B30-homoserine Insulin Precursor Using Fusion Protein
김종식†, 조정우
  • 계명대학교 화학공학과, 대구 704-701
Jong-Shik Kim†, and Chung-Woo Cho
  • Department of Chemical Engineering, Keimyung University, Daegu 704-701, Korea
E-mail:
초록
HTS-융합 B30-homoserine insulin presursor 유전자 발현 및 분리·정제 연구를 수행하였다. 플라스크상에서 재조합 단백질 유전자 발현을 위한 최적 조건은 밤새 배양한 종배양액 1% (v/v)를 새로운 배지에 주입한 후 2 h 동안 배양하고 그 배양액 1% (v/v)를 새로운 배지에 주입한 후 다시 3 h 더 배양한 다음 0.05 mM 또는 0.1 mM의 IPTG를 주입하고 다시 4 h 더 배양된 경우로 나타났다. Ni2+-affinity column chromatography에서 1X elute buffer로 유출된 분획을 SDS-PAGE로 분석한 결과 9 kDa 크기의 단일 band를 나타내는 재조합 단백질이 분리·정제되었음을 확인할 수 있었다. 아미노산 분석에서 lysine을 제외한 나머지 아미노산들의 계산치와 측정치의 유사정도는 86%이었다.
For a novel anti-diabetic drug raw material, B30-homoserine insulin precursor in the recombinant Escherichia coli cells was exmined in a flask for gene expression and purification process. The optimal culture condition for gene expression was at inoculation of 1% (v/v) pre-culture grown for 2 h to a fresh LB medium; the supplementation of 0.05 ~ 0.1 mM of IPTG for gene induction after subsequent cultivation for 3 h, and further culture for 4 h. In Ni2+-affinity column chromatography, single band of 9 kDa was appeared on SDS-PAGE for the purificationof HTS-fused B30-homoserine insulin presursor. The measured and calculated values of amino acid residues were approximately 86%, with exception to lysine.
Keywords:B30-homoserine insulin precursor;gene expression;gene induction;affinity column chromatography;purification
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