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Int. J. Mol. Sci. 2010, 11(2), 745-753; doi:10.3390/ijms11020745

Bacterial Expression of Mouse Argonaute 2 for Functional and Mutational Studies

Department of Life Sciences, Second University of Naples, Via Vivaldi 43, 81100 Caserta, Italy
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Received: 13 January 2010 / Accepted: 10 February 2010 / Published: 12 February 2010
(This article belongs to the Section Biochemistry, Molecular Biology and Biophysics)
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Abstract

RNA interference (RNAi) is a post-transcriptional gene-silencing process that occurs in many eukaryotic organisms upon intracellular exposure to double-stranded RNA. Argonaute 2 (Ago2) protein is the catalytic engine of mammalian RNAi. It contains a PIWI domain that is structurally related to RNases H and possibly shares with them a two-metal-ion catalysis mechanism. Here we describe the expression in E. coli of mouse Ago2 and testing of its enzymatic activity in a RISC assay, i.e., for the ability to cleave a target RNA in a single position specified by a complementary small interfering RNA (siRNA). The results show that the enzyme can load the siRNA and cleave the complementary RNA in absence of other cellular factors, as described for human Ago2. It was also found that mutation of Arg669, a residue previously proposed to be involved in substrate and/or B metal ion binding, doesn’t affect the enzymatic activity, suggesting that this residue doesn’t belong to the active site. View Full-Text
Keywords: RNA interference; microRNA; small interfering RNA; RNA-induced silencing complex RNA interference; microRNA; small interfering RNA; RNA-induced silencing complex
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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MDPI and ACS Style

Salvatore, V.; Potenza, N.; Papa, U.; Nobile, V.; Russo, A. Bacterial Expression of Mouse Argonaute 2 for Functional and Mutational Studies. Int. J. Mol. Sci. 2010, 11, 745-753.

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