Crystallization and preliminary crystallographic studies of human RIG-I in complex with double-stranded RNA - PubMed
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Crystallization and preliminary crystallographic studies of human RIG-I in complex with double-stranded RNA
Hyunjin Moon et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2009.
Abstract
Retinoic acid inducible gene-I (RIG-I) is an essential component of the innate immune system that is responsible for the detection and elimination of invading viruses. RIG-I recognizes viral RNAs inside the cell and then initiates downstream signalling to activate the IRF-3 and NF-kappaB genes, which results in the production of type I interferons. RIG-I is composed of an N-terminal CARD domain for signalling and C-terminal helicase and repressor domains for RNA recognition. A RIG-I-RNA binding assay was performed to investigate the in vitro RIG-I-RNA binding properties. Selenomethionine-incorporated RIG-I was expressed using Escherichia coli and purified for crystallization. X-ray data were collected from RIG-I-dsRNA complex crystals to 2.8 A resolution using synchrotron radiation.
Figures
Crystals of human RIG-I (218–925)–18-mer dsRNA complex.
Human RIG-I (218–925)–18-mer dsRNA binding assay at pH 7.0 using an electrophoretic mobility shift assay. Human RIG-I proteins were mixed with 18-mer dsRNA in 100 mM HEPES pH 7.0. After incubation at room teperature for 20 min, the mixture was applied onto a 15% TBE–polyacrylamide gel and stained with ethidium bromide. The lanes are 1 kb DNA marker, 18-mer dsRNA, RIG-I protein and 18-mer dsRNA and RIG-I mixtures with RIG-I:dsRNA molar ratios of 0.12, 0.25, 0.5, 1, 2 and 4, respectively.
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