Korean Chemical Engineering Research

The Korean Institute of Chemical Engineers (한국화학공학회)
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Development of Screening Method for the Soluble Recombinant Protein using β-Lactamase as a Fusion Partner

β-Lactamase 접합 단백질 발현 시스템을 이용한 가용성 재조합 단백질 탐색 기술 개발

  • Lee, Jae-Hun (Interdisciplinary Program for Biochemical Engineering and Biotechnology, & School of Chemical and Biological Engineering, Seoul National University) ;
  • Hwang, Bum-Yeol (Interdisciplinary Program for Biochemical Engineering and Biotechnology, & School of Chemical and Biological Engineering, Seoul National University) ;
  • Kim, Byung-Gee (Interdisciplinary Program for Biochemical Engineering and Biotechnology, & School of Chemical and Biological Engineering, Seoul National University) ;
  • Lee, Sun-Gu (Department of Chemichal Engineering, Pusan National University)
  • 이재헌 (서울대학교 협동과정 생물화학공학전공 & 서울대학교 화학생물공학부) ;
  • 황범열 (서울대학교 협동과정 생물화학공학전공 & 서울대학교 화학생물공학부) ;
  • 김병기 (서울대학교 협동과정 생물화학공학전공 & 서울대학교 화학생물공학부) ;
  • 이선구 (부산대학교 응용화학공학부)
  • Received : 2009.06.01
  • Accepted : 2009.08.18
  • Published : 2009.10.31
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Abstract

It is the most important step to screen soluble and insoluble proteins when we attempt to improve the solubility of recombinant proteins through directed evolution approach. Here we show that the solubility of a recombinant protein in vivo can be examined by expressing the recombinant protein with beta-lactamase as a fusion partner. First we constructed an expression system which can produc a fusion protein with the C-terminal of beta-lactamase. Two soluble proteins, i.e. adenine deaminase and aspartate aminotransferase, and insoluble GlcNAc-2-epimerase were cloned into the developed expression vector, respectively. We investigated the effect of the expression of the three recombinant fusion proteins on the growth of E. coli, and confirmed that the solubilities of the recombinant proteins correlated with cell growth rates.

분자진화방법을 이용하여 불용성 단백질을 가용성 단백질로 개량하고자 할 때 가장 중요한 과정은 발현 단백질의 세포 내 폴딩 및 용해도를 어떻게 측정하고 선별할 수 있는가에 있다. 본 연구에서는 ampicillin에 저항성을 가지는 beta-lactamase를 목적 단백질과 접합 형태로 발현하여 목적 단백질의 용해도를 측정 및 선별할 수 있는 방법을 구축하였다. 이를 위하여 먼저 beta-lactamase C-말단에 목적 단백질을 링커를 이용하여 접합단백질 형태로 발현시킬 수 있는 발현 시스템을 구축하였고, 구축된 발현시스템이 대장균의 ampicillin의 저항성을 향상시킴을 확인하였다. 구축된 발현시스템에 용해도가 비교적 높은 adenine deaminase와 aspartate aminotranseferase, 용해도가 매우 낮은 GlcNAc-2-epimerase 세가지 단백질의 유전자를 클로닝하여 Ampicillin 농도에 따라 목적 단백질의 용해도가 세포 성장에 미치는 영향을 조사하였다. Ampicillin 농도 $200{\mu}g/mL$에서 가용성 단백질인 adenine deaminase와 aspartate aminotranseferase의 접합 단백질 발현은 세포 성장을 보이는 반면, 불용성 단백질인 GlcNAc-2-epimerase 접합 단백질 발현은 세포 성장을 저해함을 확인하였다.

Keywords

Acknowledgement

Supported by : 부산대학교

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