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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
* Author to whom correspondence should be addressed.
Received: 13 January 2010; Accepted: 10 February 2010 / Published: 12 February 2010
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.
Keywords: RNA interference; microRNA; small interfering RNA; RNA-induced silencing complex
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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.
Salvatore V, Potenza N, Papa U, Nobile V, Russo A. Bacterial Expression of Mouse Argonaute 2 for Functional and Mutational Studies. International Journal of Molecular Sciences. 2010; 11(2):745-753.
Salvatore, Vincenzo; Potenza, Nicoletta; Papa, Umberto; Nobile, Valentina; Russo, Aniello. 2010. "Bacterial Expression of Mouse Argonaute 2 for Functional and Mutational Studies." Int. J. Mol. Sci. 11, no. 2: 745-753.