title: A generic protocol for the expression and purification of recombinant RNA in Escherichia coli using a tRNA scaffold.
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.
authors.affiliation: Laboratoire de Cristallographie et RMN Biologiques, Université Paris Descartes, CNRS, 4 avenue de l'Observatoire, Paris, France. luc.ponchon@univ-paris5.fr
authors.forename: Luc
authors.initials: L
authors.lastname: Ponchon
authors.forename: Geneviève
authors.initials: G
authors.lastname: Beauvais
authors.forename: Sylvie
authors.initials: S
authors.lastname: Nonin-Lecomte
authors.forename: Frédéric
authors.initials: F
authors.lastname: Dardel
mesh_terms: D001483:Base Sequence; D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D005821:Genetic Techniques; D005822:Genetic Vectors; D007554:Isotopes; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D012329:RNA, Bacterial / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics; D016914:Ribonuclease H
chemical_list: D007554:Isotopes; D012329:RNA, Bacterial; C039919:RNA, recombinant; D012313:RNA; D012343:RNA, Transfer; D016914:Ribonuclease H