Bacterial protein expression and purification (CaM/KRAS 1–185/BRAF RBD/SoScat)

E. coli BL21 CodonPlus cells were transformed with pET28 plasmids containing CaM or KRAS4b sequences and grown at 37°C to an optical density of 0.6 to 0.8. Temperature was reduced to 15°C before induction of protein expression with 0.25 mM isopropyl-β-d-thiogalactopyranoside. Cells were grown in Luria-Bertani media with 1 mM kanamycin for unlabeled protein production or M9 [biotin (1 mg/liter), thiamine (1 mg/liter), 0.3 mM CaCl₂, 1 mM kanamycin, 0.2% (w/v) glucose, trace elements (25 μM EDTA, 30.83 μM ferric chloride, 3.67 μM zinc chloride, 0.74 μM cupric chloride, 0.42 μM cobalt chloride, 1.62 μM boric acid, and 127.19 μM manganese chloride), and ¹⁵N-NH₄Cl (1 g/liter)] for uniform ¹⁵N labeling. Cells were lysed by sonication in buffer containing 50 mM tris-HCl (pH 8), 150 mM NaCl, 0.1% (v/v) NP-40, 10% (v/v) glycerol, 10 mM imidazole, 5 mM MgCl₂, 10 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, lysozyme, and deoxyribonuclease. CaM and unprocessed KRAS were purified by Ni–nitrilotriacetic acid His-tag affinity chromatography followed by gel filtration. A thorough purification protocol can be found here (72). N-terminally glutathione S-transferase (GST)–tagged B-Raf proto-oncogene serine/threonine kinase (BRAF) RAS-binding domain (RBD, residues 150 to 233) and His-tagged catalytic domain of SoS (SoS_cat, residues 564 to 1049) were cloned into pGEX-4T2 (GE Healthcare) and pET15b (Novagen/EMD Biosciences), respectively, expressed in E. coli BL21 (DE3), and purified as previously described (73).