4.2.4. Protein Purification

The bHLHLZ domain of N-Myc (amino acids 309–394) and Max (amino acids 12–93) protein sequences were cloned into pETDuet with an N-terminal 6×His. pETDuet-His-NMyc-Max was transformed and expressed in E. coli BL21 and induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 16 °C overnight. The cultures were lysed by sonication and combined for purification by immobilized metal ion chromatography (IMAC) with nickel-nitrilotriacetic acid (Ni-NTA) resin. The bound proteins were eluted from the column with 20 mM Tris, pH = 8, 500 mM NaCl, 5% glycerol, 300 mM imidazole, 5 mM βMe, and 0.1 mM PMSF. Size exclusion chromatography with Superdex 75 was used to increase the purity of the N-Myc-Max protein sample. The purified protein complex was used in subsequent BLI and MST experiments.