Affinity and ion exchange chromatographic columns

The coding regions of the mouse MRI and human MRI genes were codon-optimized for expression in E. coli (GenScript) and used as templates to subclone the coding regions into a modified pET15b vector (Novagen). Peptide truncations were generated by overlapping PCRs and validated by sequencing. Mouse MRI and human MRI proteins were then expressed in BL21(DE3) E. coli cells (Novagen), cultured in Luria Broth media at 37°C, induced at an OD600 (optical density at 600 nm) of 0.6 with 0.5 mM IPTG, and grown for 12-15 hours at 18°C. Cells were harvested, resuspended in lysis bu ffer (20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM 2-mercaptoethanol), and lysed using an EmulsiFlex-C5 homogenizer (Avestin), after which the resulting lysate was clarified by centrifugation (47,000 × g at 4°C, 40 minutes). Proteins were purified using a series of affinity and ion-exchange chromatographic columns (GE Healthcare). Following TEV protease digestion to separate the maltose-binding protein (MBP) fusion, the resulting sample was purified further by sequential ion-exchange and SEC. Protein purity was assessed by Coomassie staining of SDS-PAGE and mass spectrometry.

IFIT1-Ifit1 and IFIT3-Ifit3 proteins were expressed in BL21(DE3) E. coli cells (Novagen), cultured in Luria Broth media at 37°C, induced at an OD₆₀₀ (optical density at 600 nm) of 0.6 with 0.5 mM IPTG, and grown for 12–15 h at 18°C. Cells were resuspended in lysis buffer containing 25 mM sodium phosphate (pH 7.5), 250 mM NaCl, 5 mM 2-mercaptoethanol, lysed using an EmulsiFlex-C5 homogenizer (Avestin) and clarified by centrifugation at 47,000 × g at 4°C for 40 min. Proteins were purified using sequential affinity and ion-exchange chromatographic columns (GE Healthcare). Following TEV protease digestion of the maltose binding protein (MBP) tag, the resulting sample was purified further by ion-exchange and size exclusion chromatography. Protein purity was assessed by Coomassie staining of SDS-PAGE and mass spectrometry.