Purification of Mycobacterial Arr Enzymes

E. coli RNAP core and σ⁷⁰ were purified exactly as described previously (19 (link)). M. smegmatis and M. abscessus Arr were expressed in T7 express cells (New England Biolabs) transformed with pET28 expression vector encoding N-terminal 6×His-tagged M. smegmatis Arr or M. abscessus Arr. Expression was induced with 0.4 mM isopropyl-β-d-thiogalactopyranoside (IPTG) in exponentially growing cells, which were then incubated overnight at room temperature on an orbital shaker (150 rpm). Cells were then harvested by centrifugation and resuspended in grinding buffer (50 mm Tris-HCl [pH 7.9], 10% glycerol, 200 mM NaCl, and protease inhibitor mixture [Roche]). Cells were then lysed by sonication and debris cleared by centrifugation. Arr enzymes were then purified by HisTrap HP (Cytiva) nickel affinity chromatography, concentrated, and dialyzed into storage buffer (50 mM Tris-HCl [pH 7.9], 50% glycerol, 200 mM NaCl, and 2 mM β-mercaptoethanol).