Shuffle t7 competent cells

All experiments were performed in 50 mM Tris HCl, pH 8, and 100 mM sodium chloride. Buffer solutions were filtered and carefully degassed. All buffers and solutions were prepared with ultra-high-quality sterile water from Sigma Aldrich (St. Louis, Mo, U.S.A.). The pETHSUL vector, IPTG, ampicillin, and Ni-NTA resin were obtained from Thermo Scientific (Waltham, MA, U.S.A.). Shuffle T7 competent cells were obtained from New England Biolabs (NEB) (Ipswich, MA, U.S.A.). Lysogeny broth (LB) and super optimal broth (SOB) were from the BIO5 media facility (BIO5 Institute at the University of Arizona, Tucson, Arizona, U.S.A.). Antibodies for western blot immunoreactivity were obtained from Thermo Fisher Scientific (Waltham, MA, U.S.A.), and Coomassie blue (EZ-blue G-250) stain was obtained from Sigma Aldrich (St. Louis, Mo, U.S.A). The SUMO protease was obtained from MCLAB (San Francisco, CA, U.S.A.). The NAMPT protein generated by Northwestern University (Chicago, IL, U.S.A.) was utilized as a positive control for testing our expression system. All other reagents were analytical grade. Protein expression was monitored using both anti-Histidine and rat anti-NAMPT antibodies. The anti-hexa-His antibody, mouse monoclonal MA1–21315, is from ThermoFisher. The goat polyclonal anti-NAMPT antibody was custom generated as described [41 (link)].