Regeneration of ATP through an Activated Glycolytic Pathway in a Cell-free Extract and its Application for Protein Expression

해당과정의 활성화를 통한 무세포 단백질 발현 시스템에서의 ATP 재생

  • Kim Dong Myung (Department of Fine Chemical Engineering and Chemistry, Chungnam National University) ;
  • Keum Jeong Won (Interdisciplinary Programs for Biochemical Engineering and Biotechnology, Seoul National University) ;
  • Kim Tae Wan (Interdisciplinary Programs for Biochemical Engineering and Biotechnology, Seoul National University) ;
  • Oh In Seok (Interdisciplinary Programs for Biochemical Engineering and Biotechnology, Seoul National University) ;
  • Choi Cha-yong (Interdisciplinary Programs for Biochemical Engineering and Biotechnology, Seoul National University)
  • 김동명 (충남대학교 신소재공학부 정밀공업화학과) ;
  • 금정원 (서울대학교 응용화학부) ;
  • 김태완 (서울대학교 응용화학부) ;
  • 오인석 (서울대학교 응용화학부) ;
  • 최차용 (서울대학교 응용화학부)
  • Published : 2004.12.01

Abstract

We have investigated the key parameters affecting ATP regeneration in a cell-free protein synthesis system derived from Escherichia coli. When glucose-6-phosphate was used as an energy source, the efficiency of ATP regeneration sharply responded to pH change of reaction mixture. In addition, both productivity and reproducibility of protein synthesis was substantially enhanced by introducing appropriate amount of NAD into the reaction mixture. As a result, through the activation of glycolytic pathway under an optimal pH, the batch cell-free system produced over $300\;{\mu}g$ of protein in a 1 mL reaction.

해당 작용의 중간체를 에너지원으로 이용한 무세포 단백질 발현 반응에서의 낮은 재현성 및 단백질 생산성은 반응액의 pH 및 NAD의 존재에 의해 크게 영향을 받는다는 사실을 밝혀내었다. 기존의 PEP를 사용하는 표준반응 용액에서 PEP를 G-6-P로 대체하고 동시에 반응액의 pH 및 NAD 농도를 최적화 함으로써 반응액 1 mL당 약 $300{\mu}g$에 이르는 단백질을 회분식 반응으로 발현할 수 있었다. ATP 재생 방법의 개선을 통한 회분식 무세포 단백질 발현의 생산성 향상은 다종 유전자의 고속 번역을 통한 기능 규명에 있어서 유용한 도구로서 사용될 수 있을 것으로 기대된다.

Keywords

References

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