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Overexpression and Purification of Reverse Transcriptase of Retron EC83 by Changing the Downstream Sequence of the Initiation Codon
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  • Overexpression and Purification of Reverse Transcriptase of Retron EC83 by Changing the Downstream Sequence of the Initiation Codon
  • Overexpression and Purification of Reverse Transcriptase of Retron EC83 by Changing the Downstream Sequence of the Initiation Codon
저자명
JEONG. DAE-WON,LIM. DONGBIN
간행물명
Journal of microbiology and biotechnology
권/호정보
2004년|14권 6호|pp.1280-1285 (6 pages)
발행정보
한국미생물생명공학회
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정기간행물|ENG|
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이 논문은 한국과학기술정보연구원과 논문 연계를 통해 무료로 제공되는 원문입니다.
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기타언어초록

Retron is a prokaryotic genetic element, producing a short single-stranded DNA covalently linked to RNA (msDNA-RNA) by a reverse transcriptase (RT). In retron EC83, msDNA is further processed at between the 4th and the $5^{th}$ nucleotides, leaving a 79 nucleotide-long single-stranded DNA as a final product. To investigate this site-specific cleavage in msDNA synthesis, we purified the RT protein of retron EC83. Initially, RT ORF was cloned under the tac promoter, but the expression was very poor largely because of poor translation. In order to facilitate translation, the nucleotide sequence for the first nine amino acids was randomized with synonymous codons. This change of downstream sequence of translational initiation codon greatly affected the efficiency of translation. We could isolate clones which greatly increased RT production, and their sequences were compared to those of the low producers. The overproduced protein was purified and was shown to have RT activity.