Immobilized metal affinity chromatography

Genes encoding the full-length human RASSF5 isoform D (hRASSF5, UniProtKB Q8WWW0-2), the full-length human MOB1A (UniProtKB Q9H8S9-1) with two site specific substitutions T12A and T35A (hMOB1A^T2A), or the SARAH domain (residues 531-608) of Salvador from Drosophila melanogaster (dSAV-SARAH)(UniProtKB Q9VCR6)were cloned into a modified pBAT vector downstream from H6 and SUMO tags^{49 (link)}. Each of the three binding proteins were purified using the same basic protocol, as previously described^{16 (link)}. Each protein was expressed in T7 Express cells (New England BioLabs, MA) grown in Terrific Broth at 37°C following induction with 0.5mM IPTG. Cells were lysed in 50mM Tris pH 8, 400mM NaCl, 10% glycerol. Proteins were purified by Nickel charged Immobilized Metal Affinity Chromatography (BioRad, CA). The affinity tags were removed during overnight incubation with SENP (made in-house), and the untagged protein further purified by gel-filtration chromatography, concentrated to a minimum of 100μM, and flash frozen in liquid nitrogen. Modifications to this protocol included purification and concentration of each protein prior to gel-filtration. hRASSF5 was concentrated by 45% w/v ammonium sulfate precipitation; hMOB1A^T2A was purified on anion-exchange chromatography; dSav-SARAH was purified by anion exchange chromatography and then concentrated by 60% w/v ammonium sulfate precipitation.