Expression,Protein Purification,Crystallization Of The Viral RNA Receptor RIG-I

In eukaryotic cells,Toll-like receptors in endosomal membrane were show to recognize viral RNA in endosome and thereby initiate innate immunity pathways for virus resistance.By contrast,a cytosolic RNA receptor protein, RIG-Ⅰ(retinoic acid inducible gene-Ⅰ),has recently been identified for recognition of the viral RNA.RIG-Ⅰsignals to its downstream adaptor protein MAVS(mitochondrial anti-viral signaling protein) through the CARD-CARD interaction and subsequently activates the transcription factors IRF-3 and NF-κB.Once activated,these two transcription factors translocate into the nucleus for inducing expression of INF-β,thus resulting in initiation of innate immune response and subsequent regulation of acquired immune response. RIG-Ⅰcontains C-terminal RNA helicase domain and two tandem CARD domains at N-terminus.Under normal conditions,the C-terminal helicase domain inhibits the signaling function of N-terminal CARD domain,which can be relieved by recognition of the viral RNA by RIG-Ⅰ.The mechanism underlying this process is believed to be through conformational changes induced by the binding of viral RNA and thereby exposing the CARD domains of RIG-Ⅰ.However,structural evidence for this is lacking.Our thesis starts with purification of large amount of recombinant RIG-Ⅰprotein suitable for crystallization.By co-expression,we found that the C-terminal RNA helicase domain interacted with the N-terminal CARD domain of RIG-Ⅰ,providing a biochemical evidence for the auto-inhibition of RIG-Ⅰ.Consistents with previous results, Electrophoretic Mobility Shift Assays,EMSA,showed that the in vitro transcribed dsRNA or ssRNA of 5'-triphosphate and the chemical synthesized dsRNA bearing no 5'-triphosphate can be recognized by RIG-Ⅰ.Using the elutrap or dialyzer,we purified various RIG-Ⅰ/RNA complexes for crystallization.Like RIG-Ⅰitself,all these protein-RNA complexes are refractory for crystallization.