IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA

Renata C Fleith; Harriet V Mears; Edward Emmott; Stephen C Graham; Daniel S Mansur; Trevor R Sweeney

For mammalian cell expression, sequences for human IFIT1 (BC007091.1) and IFIT5 (BC025786.1) were PCR amplified to include a 5' Kozak sequence, 3' FLAG tag and 5' BamHI and 3' XhoI sites, to facilitate cloning into pCDNA3.1. The plasmid for expression of IFIT1 in Escherichia coli was previously described (10) and was used as a template for site directed mutagenesis to generate the mutant IFIT1 expression vectors. Sequences for IFIT2 (NM_001547.4) and IFIT3 (NM_001549.5) were IFIT2 and IFIT3 were further purified on MonoQ 5/50 (Q buffer), followed by size exclusion chromatography (SEC) on Superdex 200 increase 10/300 GL or HiLoad 16/600 Superdex 200 pg columns (SEC buffer: 20 mM Tris-Cl pH 7.5, 150 mM KCl and 1 mM DTT). All FPLC columns are from GE Healthcare and all buffer reagents and enzymes were from Sigma.