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A generic protocol for the expression and purification of recombinant RNA in Escherichia coli using a tRNA scaffold

Nature Protocols volume 4, pages 947–959 (2009)Cite this article

Abstract

RNA production using in vivo transcription by Escherichia coli allows preparation of milligram quantities of RNA for biochemical, biophysical and structural investigations. We describe here a generic protocol for the overproduction and purification of recombinant RNA using liquid chromatography. The strategy utilizes a transfer RNA (tRNA) as a scaffold that can be removed from the RNA of interest by digestion of the fusion RNA at a designed site by RNase H. The tRNA scaffold serves to enhance the stability and to promote the proper expression of its fusion partners. This protocol describes how to construct a tRNA fusion RNA expression vector; to conduct a pilot experiment to assess the yield of the recombinant RNA both before and after processing of the fusion RNA by RNase H; and to purify the target RNA on a large scale for structural or functional studies. This protocol greatly facilitates production of RNA in a time frame of ∼3 weeks from design to purification. As compared with in vitro methods (transcription, chemical synthesis), this approach is simple, cheap and well suited for large-scale expression and isotope labeling.

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Figure 1: General scheme for the construction of tRNA chimera expressing vectors.
Figure 2: Circular maps of the pBSKrna plasmid and structure of the inserts of the various tRNA expression vectors, which are derivatives of pBSTNAV.
Figure 3: Gel images showing the RNA chimeras at various stages of the protocol.
Figure 4: The different cleavage steps carried out by RNase H.
Figure 5: Purification of RNase H cleavage products by ion-exchange chromatography under denaturing conditions (4 M urea).

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References

  1. Marshall, W.S. & Kaiser, R.J. Recent advances in the high-speed solid phase synthesis of RNA. Curr. Opin. Chem. Biol. 8, 222–229 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Schenborn, E.T. & Mierendorf, R.C. Jr. A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure. Nucleic Acids Res. 16, 6223–6236 (1985).

    Article  Google Scholar 

  3. Lukavsky, P.J. & Puglisi, J.D. Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides. RNA 10, 889–893 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Cheong, H.K., Hwang, E., Lee, C., Choi, B.S. & Cheong, C. Rapid preparation of RNA samples for NMR spectroscopy and X-ray crystallography. Nucleic Acids Res. 32, e84 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Kieft, J.S. & Batey, R.T. A general method for rapid and nondenaturing purification of RNAs. RNA 10, 988–995 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Mayer, M. & James, T.L. NMR-based characterisation of phenothiazines as a RNA binding scaffold. J. Am. Chem. Soc. 126, 4453–4460 (2004).

    Article  CAS  PubMed  Google Scholar 

  7. Masson, J.-M & Miller, J.H. Expression of synthetic tRNA genes under the control of a synthetic promoter. Gene 47, 179–183 (1986).

    Article  CAS  PubMed  Google Scholar 

  8. Meinnel, T., Mechulam, Y. & Fayat, G. Fast purification of a functional elongator tRNAmet expressed from a synthetic gene in vivo . Nucleic Acids Res. 16, 8095–8096 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Tisné, C., Rigourd, M., Marquet, R., Ehresmann, C. & Dardel, F. NMR and biochemical characterization of recombinant human tRNA(Lys)3 expressed in Escherichia coli: identification of posttranscriptional nucleotide modifications required for efficient initiation of HIV-1 reverse transcription. RNA 6, 1403–1412 (2000).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Wallis, N.G., Dardel, F. & Blanquet, S. Heteronuclear NMR studies of the interactions of 15N-labeled methionine-specific transfer RNAs with methionyl-tRNA transformylase. Biochemistry 34, 7668–7677 (1995).

    Article  CAS  PubMed  Google Scholar 

  11. Deutscher, M.P. Ribonucleases, tRNA nucleotidyltransferase, and the 3′ processing of tRNA. Prog. Nucleic Acid Res. Mol. Biol. 39, 209–240 (1990).

    Article  CAS  PubMed  Google Scholar 

  12. Engelke, D.R. & Hopper, A.K. Modified view of tRNA: stability amid sequence diversity. Mol. Cell 21, 144–145 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Ponchon, L. & Dardel, F. Recombinant RNA technology: the tRNA scaffold. Nat. Methods 4, 571–576 (2007).

    Article  CAS  PubMed  Google Scholar 

  14. Smith, D.B. & Johnson, K.S. Single-step purification of polypeptides expressed in E. coli as fusions with glutathione S-transferase. Gene 67, 31–40 (1988).

    Article  CAS  Google Scholar 

  15. McKenna, S.A. et al. Purification and characterization of transcribed RNAs using gel filtration chromatography. Nat. Protoc. 2, 3270–3277 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Nassal, M. Hepatitis B viruses: reverse transcription a different way. Virus Res. 134, 235–249 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd edn. (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989).

    Google Scholar 

  19. Gaudin, C. et al. The tRNA-like domains of E. coli and A. aeolicus transfer-messenger RNA: structural and functional studies. J. Mol. Biol. 331, 457–471 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

    Article  CAS  Google Scholar 

  21. Fourmy, D., Recht, M.I., Blanchard, S.C. & Puglisi, J.D. Structure of the A site of E. coli 16 ribosomal RNA complexed with an aminoglycoside antibiotic. Science 274, 1367–1371 (1996).

    Article  CAS  PubMed  Google Scholar 

  22. Fürtig, B., Richter, C., Wöhnert, J. & Schwalbe, H. NMR spectroscopy of RNA. Chembiochem. 4, 936–962 (2003).

    Article  PubMed  Google Scholar 

  23. Schumacher, J., Meyer, N., Riesner, D. & Weidemann, H.L. Diagnostic procedure for detection of viroids and viruses with circular RNAs by 'Return'-gel electrophoresis,. J. Phytopathol. 115, 332–343 (1986).

    Article  Google Scholar 

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Authors and Affiliations

  1. Laboratoire de Cristallographie et RMN Biologiques, Université Paris Descartes, CNRS, 4 avenue de l'Observatoire, Paris, France

    Luc Ponchon, Geneviève Beauvais, Sylvie Nonin-Lecomte & Frédéric Dardel

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  1. Luc Ponchon
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  2. Geneviève Beauvais
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  3. Sylvie Nonin-Lecomte
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  4. Frédéric Dardel
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Correspondence to Luc Ponchon.

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Ponchon, L., Beauvais, G., Nonin-Lecomte, S. et al. A generic protocol for the expression and purification of recombinant RNA in Escherichia coli using a tRNA scaffold. Nat Protoc 4, 947–959 (2009). https://doi.org/10.1038/nprot.2009.67

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