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KMID : 0545120040140061280
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Journal of Microbiology and Biotechnology
2004 Volume.14 No. 6 p.1280 ~ p.1285
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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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Jeong DW
Lim DB
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Abstract
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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 5th 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.
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