Heterologous Expression and Purification of ELIC

The pET-26-MBP-ELIC was provided by Raimund Dutzler (Addgene plasmid # 39239) and was used for the generation of both WT and mutant ELIC. Site-directed mutagenesis was conducted using the Quikchange method and subsequently confirmed by Sanger sequencing (Genewiz). WT and mutant ELIC were expressed as previously reported^{15 (link),54 (link)} in OverExpress C43 (DE3) E. Coli (Lucigen). Terrific Broth (Sigma) was used to grow cultures which were induced with 0.1 mM IPTG for ~16 h at 18 °C. The cells were pelleted, resuspended in Buffer A (20 mM Tris pH 7.5, 100 mM NaCl) with complete EDTA-free protease inhibitor (Roche), and lysed using an Avestin C5 emulsifier at ~15,000 psi. Membranes were pelleted by ultracentrifugation, resuspended in Buffer A, solubilized with 1% DDM (Anatrace), and incubated with amylose resin (New England Biolabs) for 2 h. The amylose resin was washed with 20 bed volumes of Buffer A containing 0.02% DDM and 0.05 mM TCEP (ThermoFisher Scientific), and eluted with 40 mM maltose (Sigma). The eluted protein was digested with HRV-3C protease (ThermoFisher Scientific) (10 units per mg ELIC) overnight at 4 °C then purified over a Sephadex 200 Increase 10/300 (GE Healthcare Life Sciences) size exclusion column in Buffer A with 0.02% DDM.