Protein X was expressed in E. coli from a plasmid under the control of a T7 promoter and the gene of interest.
A 100 mL overnight culture was grown at 37ºC. To scale up, 16 hours later a 10 mL aliquot of the overnight culture was added to each of eight, 2 liter baffled flasks. Four flasks contained 1 L of (Luria Bertani) LB media and four flasks contained 1 L of a super rich media that anecdotal data had shown improves expression of Protein X (50 mM Tris pH 7.5, 10 g yeast extract, 25 g tryptone, 5 g glucose, 5 g MgSO4).
All eight cultures were grown at 30ºC and were shaken at 200 rpm until O.D. 600 reached 0.5 (approximately three hours). At this point Isopropyl ß-D-1-thiogalactopyranoside (IPTG) was added to each flask to a final concentration of 200 µM. IPTG is a gratuitous inducer of the lac promoter, which is used to drive T7 polymerase expression in DE3 cells. The Protein X gene has a T7 promoter and is transcribed by T7 polymerase, allowing it to be overexpressed in the cell.
At this point, two LB and two super rich flasks were moved to a second shaker running at 15ºC and 200 rpm. The remaining four flasks were kept in the original shaker, at 200 rpm, but its temperature was lowered to 25ºC. (Flasks equilibrated to their new temperatures in less than 30 minutes, at this time-point protein expression of our gene of interest in undetectable).
At three hours, the cells from one flask at each condition (15ºC LB; 15ºC super rich, 25ºC LB; 25 ºC super rich) were harvested by centrifugation (four minutes @ 12,000 xg). The remaining four flasks were harvested the same way 16 hours after induction. Cell pellets were all frozen at -80ºC to aid lysis.
Cells were lysed in Thermo Scientific B-PER Bacterial Protein Extraction Reagent and enzymes and the lysate cleared by centrifugation (20 minutes @ 48,000 xg). Each lysate was then purified on a fresh 1 mL Thermo Scientific HisPur Cobalt Resin column using fast protein liquid chromatography at a flow rate of 2 mL/min. The column was washed with 20 mM Tris (pH 8.0 500 mM NaCl). Increasing concentrations of imidazole (10 mM, 20 mM, and 250 mM) were used to wash and then elute Protein X.
Fractions were analyzed by SDS gel electrophoresis (Figure 1) and quantitated by densitometry to determine the amount of protein X that had been purified. Measurements were performed in duplicate to assess error. The data was then analyzed statistically using the Design-Expert® 7 Workstation.