Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Evaluation of Novel Constitutive Expression Vectors Equipped with Mined Promoters from Metagenome

메타게놈에서 발굴한 프로모터를 장착한 새로운 항시발현 벡터의 가치평가

  • Han, Sang-Soo (Department of Biological Sciences, College of Natural Science, Chonnam National University) ;
  • Kim, Geun-Joong (Department of Biological Sciences, College of Natural Science, Chonnam National University)
  • 한상수 (전남대학교 분자생명과학부 생물학과) ;
  • 김근중 (전남대학교 분자생명과학부 생물학과)
  • Published : 2008.12.28
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Abstract

The choice of expression vector is very important for industrial production of proteins. Therefore, the systematic mining of promoters over a wider range of genetic resource and/or host is required. We previously reported a novel bidirectional reporting system (pBGR) for the isolation of promoters from metagenome and screened useful promoters that functioned constitutively in E. coli under general culture conditions. Among them, three promoter sequences including each upstream region were amplified by PCR and used to construct new expression vectors. To facilitate subcloning, a multi-cloning site was incorporated into the downstream region of the revere primer sequence. At these sites, GFP, esterase and $\beta$-glucosidase were subcloned and analyzed the constitutive expression ability of new promoter in terms of protein solubility and expression level. As a result, these vectors expressed the proteins constitutively to a level of $2{\sim}3%$ of the total cell protein in soluble fraction (>80 %). This study suggested that excavation of metagenomic promoters for construction of expression vector in a certain strain could provide a way for the development of the expression systems.

단백질의 산업적 생산을 위해 발현벡터의 선정이 중요하지만 이용 가능한 프로모터가 극히 제한적이며 많은 경우 과발현되는 특성과 함께 불용성 응집체가 형성되는 단점을 지닌다. 따라서 다양한 생물로부터 유래된 잠재성이 큰 유전자원(metagenome)에서의 프로모터 발굴과 한정된 숙주를 해결하려는 노력이 요구된다. 선행연구에서 발굴한 metagenome 유래의 항시발현 프로모터를 이용해 대장균의 일반적인 배양조건에서 세포생리에 영향이 적은 신규 항시발현 벡터를 제작하였다. 이를 위해 예측된 프로모터 서열과 MCS를 포함하는 합성 primer를 제작한 후 PCR로 증폭해 발현벡터를 구성한 후, 프로모터 구동여부와 단백질 발현양상 등을 관찰하였다. 인위적으로 도입된 MCS에 GFP, esterase, $\beta$-glucosidase를 클로닝해 단백질 발현양과 가용성을 분석한 결과, 안정적으로 전체단백질의 $2{\sim}3%$ 정도로 발현되며 80% 이상의 높은 가용성을 지닌 단백질의 발현이 유도되는 것으로 확인되었다. 이와 같은 결과는 잠재적인 생물자원의 보고로서 metagenome의 활용가능성을 제시하고 있다. 따라서 다양한 숙주에서 작동하는 프로모터의 발굴 및 발현벡터의 제작을 시도할 경우 단백질의 생산이나 대사공학에 의한 균주개량에 유용하게 활용할 수 있을 것이다.

Keywords

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