Expressing and Purifying Bacterial Proteases

Reagent grade (or better) Tris Base (2-amino-2-(hydroxymethyl)-1,3-propanediol), calcium chloride, hydrochloric acid, dithiothreitol (DTT), imidazole, isopropyl-β-D-thiogalactopyranoside (IPTG), Nα-benzoyl-DL-arginine-4-nitroanilide (BApNA), kanamycin, Luria Broth, and Terrific Broth were purchased from Fisher Scientific (Thermo Fisher Scientific, Waltham, MA). To visualize protease expression of bacterial lysates and of concentrated purified proteases, 4–12% (gradient) or 12% NuPAGE™ Bis-Tris Protein Gels (Invitrogen, Carlsbad, CA) were used and stained with either InVision™ His-Tag In-Gel Stain (Invitrogen #LC6030), SimplyBlue™ Safe Stain (Invitrogen #LC6065) or both. For western blots, AaET-specific and other custom antibodies were purchased from GenScript (Piscataway, NJ) and have been previously described [1 (link), 15 (link)].