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Journal of microbiology and biotechnology v.14 no.6, 2004년, pp.1280 - 1285  
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Overexpression and Purification of Reverse Transcriptase of Retron EC83 by Changing the Downstream Sequence of the Initiation Codon

JEONG , DAE-WON    (BK21 HLS, Seoul National University   ); LIM, DONG-BIN    (Department of Bioinformatics and Life Science, College of Natural Sciences, Soongsil University  );
  • 초록

    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.


  • 주제어

    Retron .   reverse transcriptase .   translational control .   overexpression.  

  • 참고문헌 (21)

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    2. Kim, M. D., K.-N. Kim, and Y. J. Choi. 2003. Enhanced expression of $\beta$-xylosidase of Bacillus stearothermophilus No. 236 by change of translational inhibition codon in Escherichia coli and Bacillus subtilis. J. Microbiol. Biotechnol. 13: 584-590 
    3. Lim, D. and W. K. Maas. 1989. Reverse transcriptasedependent synthesis of a covalently linked, branched DNARNA compound in E. coli B. Cell 56: 891-904 
    4. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. U.S.A 
    5. Lim, D. and W. K. Maas. 1989. Reverse transcriptase in bacteria. Mol. Microbiol. 3: 1141-1144 
    6. Oh, K.-S., Y.-S. Park, and H.-C. Sung. 2002. Expression and purification of an ACE-inhibitory peptide multimer from synthetic DNA in Escherichia coli. J. Microbiol. Biotechnol. 12: 59-64 
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    10. Kuroda, H. and P. Maliga. 2001. Sequences downstream of the translation initiation codon are important determinants of translation efficiency in chloroplasts. Plant Physiol. 125: 430-436 
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    12. Kim, K., D. W. Jeong, and D. Lim. 1997. A mutational study of the site-specific cleavage of EC83, a multicopy singlestranded DNA (msDNA): Nucleotides at the msDNA stem are important for its cleavage. J. Bacteriol. 179: 6518- 6521 
    13. Jeong, D. W., K. Kim, and D. Lim. 1997. Evidence for the complex formation between reverse transcriptase and multicopy single-stranded DNA in retron EC83. Mol. Cells 7: 347-351 
    14. Lim, D. 1992. Structure and biosynthesis of unbranched multicopy single-stranded DNA by reverse transcriptase in a clinical Escherichia coli isolate. Mol. Microbiol. 6: 3531- 3542 
    15. Temin, H. M. 1989. Retrons in bacteria. Nature 339: 254- 255 
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    17. Maas, W. K., C. Wang, T. Lima, G. Zubay, and D. Lim. 1994. Multicopy single-stranded DNAs with mismatched base pairs are mutagenic in Escherichia coli. Mol. Microbiol. 14: 437-441 
    18. Sprengart, M. L., E. Fuchs, and A. G. Porter. 1996. The downstream box: An efficient and independent translation initiation signal in Escherichia coli. EMBO J. 15: 665-674 
    19. Etchegaray, J. P. and M. Inouye. 1999. A sequence downstream of the initiation codon is essential for cold shock induction of cspB of Escherichia coli. J. Bacteriol. 181: 5852-5854 
    20. Oh, Y. P., S. T. Jeong, D.-W. Kim, E.-C. Kim, and K.-H. Yoon. 2002. Simple purification of shiga toxin B chain from recombinant Escherichia coli. J. Microbiol. Biotechnol. 12: 986-988 
    21. Kim, J. S., H. Y. Seong, M. W. Kim, J. S. Ku, and S. Y. Choi. 2003. Effects of minor arginyl tRNA and isoleucyl tRNA on the expression of Clostridium botulinum neurotoxin light chain in Escherichia coli. J. Microbiol. Biotechnol. 13: 287- 291 

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