E. coli expression and purification of hINMT proteins (254C, 254F, D28N, H46P, M206V, N245S)

N-terminally His-tagged human INMT proteins were expressed, amplified and batch-purified using standard purification procedures in the absence of reducing agent. Miss-sense mutations were created using the Quickchange method (Aligent) on pet28a vector encoding ‘WT’ (254C) hINMT (S2 Fig) and verified by sequencing. All hINMT genes were N-terminally His-tagged to enable cobalt bead purification. E-coli BL-21 (Lucigen, Madison) cells were transformed with pet28a vector encoding N-terminally His-tagged hINMT construct. Following pre-culture, transformed BL-21 cells were grown to OD₆₀₀ = 600 at 37° in 6 L Luria broth (LB) under kanamycin (50 μg/ml) selection at which point hINMT expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cultures grown an additional 3 hours. Bacteria were collected by centrifugation and resuspended in 25 ml buffer A (50 mM tris-HCl pH 7.5, 150 mM NaCl, protease inhibitor cocktail [Dot Scientific, Burton]). The suspension was sonicated eight times in 30 sec. on/off cycles at 4°C at output 4. Sonicated membranes were pelleted by centrifugation (13,000 x G, 20 min). 500 μl of buffer A pre-equilibrated HisPure cobalt resin (ThermoFisher Scientific, Waltham) and 20 mM imidazole were added to the lysate and rotated for 1 hr at ambient temperature. The resin was washed 3X in 2 ml buffer B (50 mM NaH₂PO₄ pH 7.8, 20 mM imidazole) and eluted with 2X 3 ml buffer B incubations with 300 mM imidazole. Eluted volumes were combined and dialyzed twice overnight in 6 L of buffer B at 4°C to remove imidazole. The dialyzed proteins were concentrated to approximately 1 mg/ml using a Vivaspin 6 column (Sartorius, Goettingen) and verified by acrylamide gel Coomassie staining for molecular size and purity. Pure protein yield was generally in the 2–3 mg range. Protein was adjusted to a final concentration of ca. 0.5 mg/ml and stored in 50–90 μl aliquots at -80°C in 20% glycerol, 40 mM NaH₂PO₄ pH 7.5; 130 mM NaCl until use. For added comparison, pure protein samples of the hINMT allozymes WT (254C), D28N, H46P, M206V, G219E and 254F are shown in the presence of reducing agent in S3 Fig.