Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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In vitro Synthesis of Ribonucleic Acids by T7 RNA Polymerase That was Fast Purified with a Modified Procedure

변형된 방법으로 신속히 정제된 T7 RNA 중합효소를 이용한 리보핵산의 시험관 내 합성

  • Kim Ki-Sun (Department of Biotechnology and Bioengineering, College of Engineering, Dong-Eui University) ;
  • Choi Woo-Hyung (Department of Biotechnology and Bioengineering,College of Engineering, Dong-Eui University) ;
  • Gong Soo-Jung (Department of Biotechnology and Bioengineering,College of Engineering, Dong-Eui University) ;
  • Jeon Sung-Jong (Department of Biotechnology and Bioengineering, Department of Biomaterial Control,College of Engineering, Dong-Eui University) ;
  • Kim Jae Hyun (Nambu Blood Laboratory, Korean Red Cross) ;
  • Oh Sangtaek (PharmcoGenomics Research Center, Inje Ufiversity) ;
  • Kim Dong-Eun (Department of Biotechnology and Bioengineering, Department of Biomaterial Control,College of Engineering, Dong-Eui University)
  • 김기선 (동의대학교 공과대학 생명공학과) ;
  • 최우형 (동의대학교 공과대학 생명공학과) ;
  • 공수정 (동의대학교 공과대학 생명공학과) ;
  • 전숭종 (동의대학교 공과대학 생명공학과, 동의대학교 대학원 바이오물질제어학과) ;
  • 김재현 (대한적십자 남부 혈액검사센터) ;
  • 오상택 (인제대학교 약물유전체 연구센터) ;
  • 김동은 (동의대학교 공과대학 생명공학과, 동의대학교 대학원 바이오물질제어학과)
  • Published : 2005.10.01
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Abstract

Biochemical amounts of RNA molecules can be synthesized in vitro, which is functionally equivalent or similar to those transcripts normally existing at extremely low levels in vivo. In this study we described a method for efficient preparation of pure T7 RNA polymerase from Escherichia coli strain BL21/pAR1219. The procedure, which used ammonium sulfate fractionation and preparative column chromatography on sephadex SP, was shown to be simple, rapid, and cost effective in comparison with other methods reported previously, Using the purified T7 RNA polymerase we were able to synthesize very long RNA transcript of 1.54 kb length, which is not feasible by conventional chemical synthesis. RNA molecule that was also synthesized by the purified T7 RNA polymerase, such as hammerhead ribozyme, retained its biochemical activity by cleaving the target RNA successfully in vitro. Thus, the procedure shown in this study can be useful to synthesize any length of RNA molecules in vitro in a simple and cost effective way for a variety of purposes.

세포 내에서 매우 적은 양으로 존재하는 RNA 전사체와 기능적으로 동일하거나 비슷한 RNA를 RNA 중합효소를 써서 in vitro에서 생화학적으로 의미 있는 양 만큼을 합성할 수 있다. T7 RNA중합효소를 발현하는 재조합 유전자를 지닌 대장균주 BL21/pAR1219로부터 순수한 T7 RNA중합효소를 손쉽게 얻는 방법을 본 논문에서 소개한다. 황산암모늄 분획화와 sephadex SP 컬럼 크로마토그래피법으로써 여타의 방법과 비교하여 더 간단하고 빠르게, 그리고 경제적으로 T7 RNA 중합효소를 분리할 수 있었다. 정제된 T7 RNA중합효소를 이용하여 보통의 화학적 합성법으로 불가능한 긴 길이(1.54 kb)의 RNA전사체를 합성 하였다. 한편,정제된 T7 RNA중합효소에 의해 생성된 망치머리 리보자임은 표적 RNA를 in vitro에서 절단함으로써, 생성된 RNA가 생화학적 기능성을 유지한다는 것을 입증하였다 따라서 본 연구에서 소개되는 절차들은 다양한 길이의 RNA를 목적에 따라 간단하고 경제적으로 합성하는데 유용하게 이용될 수 있다.

Keywords

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