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Nat Protoc. 2009;4(6):947-59. doi: 10.1038/nprot.2009.67. Epub 2009 May 28.

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

Author information

1
Laboratoire de Cristallographie et RMN Biologiques, Université Paris Descartes, CNRS, 4 avenue de l'Observatoire, Paris, France. luc.ponchon@univ-paris5.fr

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 approximately 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.

PMID:
19478810
DOI:
10.1038/nprot.2009.67
[Indexed for MEDLINE]

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