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.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Ponchon L,Beauvais G,Nonin-Lecomte S,Dardel Fdoi
10.1038/nprot.2009.67subject
Has Abstractpub_date
2009-01-01 00:00:00pages
947-59issue
6eissn
1754-2189issn
1750-2799pii
nprot.2009.67journal_volume
4pub_type
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