Purification of Recombinant NAMPT Protein

Recombinant NAMPT protein was purified as we previously described^{9 (link), 10 (link)}. In brief, human NAMPT cDNA tagged with His6 at the C-terminus was inserted into pET-30a (Novagen, Madison, Wisconsin) and transformed into E. Coli BL21. Recombinant protein was induced with isopropyl β-D-1-thiogalactopyranoside (0.1 mM) at 25 °C for 6 hr. Recombinant protein was purified using the Ni-NTA Fast Start Kit (Qiagen, Germantown, MD) and dialyzed sequentially in 300 mM NaCl/10 mM imidazole and 300 mM NaCl/10 mM Tris-HCl (pH 8.0). To remove endotoxins, purified protein was passed through Detoxi-Gel Endotoxin Removing Columns as per the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA). A NAD enzyme-inactive NAMPT with the H247A point mutation was generated with the same procedures as above. The purified protein was filtered through a 0.2-μm filter, aliquoted, and stored at −70 °C. Purified proteins were verified by Coomassie blue staining and Western blot analyses, and protein concentrations were determined with the Pierce BCA Protein Assay (Thermo Fisher Scientific).