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

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


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.

Publication types

  • Comparative Study
  • Evaluation Study

MeSH terms

  • Base Sequence
  • Chromatography, Liquid / methods
  • Escherichia coli / genetics*
  • Genetic Techniques*
  • Genetic Vectors
  • Isotopes
  • Molecular Sequence Data
  • Plasmids / genetics
  • RNA / chemistry
  • RNA / genetics*
  • RNA / isolation & purification*
  • RNA, Bacterial / genetics
  • RNA, Transfer / genetics
  • Ribonuclease H


  • Isotopes
  • RNA, Bacterial
  • RNA, recombinant
  • RNA
  • RNA, Transfer
  • Ribonuclease H