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

• Published in: Nature protocols Vol. 4; no. 6; pp. 947 - 959
• Main Authors: Ponchon, Luc, Beauvais, Geneviève, Nonin-Lecomte, Sylvie, Dardel, Frédéric
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.06.2009; Nature Publishing Group
• Subjects: Analytical Chemistry; Base Sequence; Biochemistry, Molecular Biology; Biological Techniques; Biomedical and Life Sciences; Chromatography, Liquid - methods; Computational Biology/Bioinformatics; E coli; Escherichia coli; Escherichia coli - genetics; Genetic aspects; Genetic research; Genetic Techniques; Genetic Vectors; Isotopes; Life Sciences; Liquid chromatography; Methods; Microarrays; Molecular biology; Molecular Sequence Data; Organic Chemistry; Physiological aspects; Plasmids - genetics; Properties; protocol; Ribonuclease H; RNA; RNA - chemistry; RNA - genetics; RNA - isolation & purification; RNA processing; RNA, Bacterial - genetics; RNA, Transfer - genetics; Structural Biology; Transfer RNA; France
• ISSN: 1754-2189; 1750-2799; 1750-2799
• DOI: 10.1038/nprot.2009.67

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.