Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Overexpression of the get Gene Encoding 4-α-Glucanotransferase of a Hyperthermophilic Archaeon, Thermococcus litoralis

초호열성 고세균 Thermococcus litoralis로부터 4-α-glucanotransferase의 대량밭현

  • Jeon, Beong-Sam (Institute of Life Science, Gyeongsang National University) ;
  • Park, Jeong-Won (Institute of Life Science, Gyeongsang National University) ;
  • Shin, Gab-Gyun (Institute of Life Science, Gyeongsang National University) ;
  • Kim, Beom-Kyu (Institute of Life Science, Gyeongsang National University) ;
  • Kim, Hee-Kyu (Institute of Life Science, Gyeongsang National University) ;
  • Song, Jae-Young (Department of Microbiology, College of Medicine, Gyeongsang National University) ;
  • Cho, Young-Su (Department of Biotechnology, College of Natural Resources and Life Science, Dong-A University) ;
  • Cha, Jae-Young (Institute of Life Science, Gyeongsang National University)
  • Published : 2004.06.01
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Abstract

The gene encoding a extremely thermostable 4-$\alpha$-glucanotransferase from a hyperthermophilic archaeon, Thermococcus litoralis, was cloned, sequenced and expressed in Escherichia coli. The amino acid sequence of the enzyme was distantly related to other functionally-related ones, such as D-enzymes. The enzyme is of industrial interest because of a novel activity of producing cycloamylose and is also important for fundamental studies of protein, sugar-metabolizing enzymes. In this paper, the overexpression of 4-$\alpha$-glucanotransferase in E. coli was carried out expression vector system with lac and T7 promoters. The enzyme was successfully overexpressed, and purified by the heat treatment of a cell-free extract, successive Butyl-Toyopearl and Mono Q chromatographies. The purified recombinant enzyme showed the same specific activity and the same mobility in SDS-PAGE as natural enzyme.

초호열성 고세균 Thermococcus litoralis 유래의 4-$\alpha$-glucanotransferase는 클로닝 되어 염기배열이 밝혀졌으며, 대장균에서 발현되었다 발현된 이 효소는 기능적인 면에서는 D-enzyme과 유사하지만 아미노산 배열에서는 큰 차이점을 나타내었다. 이 효소는 cycloamylose를 생산하는 새로운 기능적인 특성을 가지고 있어 당대사 관련 효소 단백질에 관한 연구의 중요성 때문에 산업적으로 많은 각광을 받고 있다. 본 연구는 초호열성 고세균 T. litoris 유래 4-$\alpha$-glucanotransferase 유전자를 부위 특이적 변이 방법으로 재조합하여 lac와 T7프로모터를 이용해서 대장균 발현 벡터 시스템에서 대량발현 시켰다. 대장균에서 대량 발현된 재조합 효소 단백질은 열처리, 501빌Butyl-Toyopearl, Mono Q 크로마티그래피 방법에 의하여 간단히 정제되었다. 정제된 재조합 효소 단백질은 본래의 효소 단백질과 같은 기능을 가지고 있는 것으로 확인되었다.

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