Laboratory name
Washington University
Expression of natively-folded/soluble proteins in E. coli. Day 1. Streak an LB-carbenicillin plate from frozen stocks to obtain isolated colonies of the expression plasmid in E. coli strain BL21-CodonPlus (DE3)-RIL cells (Agilent Technologies Cat. No. 230245 ). Incubate the plate overnight at 37°C. Day 2. Seed a single colony into 1000 ml of LB with 100 µg/ml carbenicillin and shake at 225 rpm in a 37°C incubator. Grow until the culture density reaches between 0.6 and 0.8 OD600, usually about 5 hours. Collect a 5 ml sample of non-induced bacteria and store it at 4°C. Lower the temperature to 25°C. Induce protein expression with IPTG to a final concentration of 0.5 mM (120 mg per liter). Allow the culture to grow between 4 and 12 hours. Collect a 2 ml sample of induced bacteria and store it a 4°C. Pellet the bacteria by centrifugation at 5000 rpm for 20 minutes and at 4°C. The pellet can be flash frozen in liquid nitrogen and stored at -80°C until you are ready to proceed with protein purification. To examine the extent of induction adjust the OD600 of the uninduced and induced samples to 1.4 with TE. Pellet 1 ml of each and dissolve in 200 µl of 2x SDS-PAGE loading buffer (125 mM Tris-HCl pH6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue). Sonicate using a microtip sonicator to break open the cells and shear the DNA. Be careful not to foam the samples while sonicating. Load 20 µl/lane of each sample onto a 10% SDS-PAGE with molecular weight protein standards. Run the gel and stain with Imperial Protein Stain (Pierce Cat. No.24615) to compare the uninduced and induced sample band intensities.