pET32a (+)-Glu-Em7

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	The recombinant strain E. coli BL21 (DE3)/pET32a (+)-Glu was inoculated in 5 mL LB liquid medium (containing 100 μg/mL ampicillin) and cultured at 37°C overnight. Then, 1 mL of this culture was inoculated into 100 mL LB medium (containing 100 μg/mL ampicillin) and grown at 37°C with shaking at 200 rpm until the OD600 value reached 0.4-0.6.

Protocol tips
The recombinant strain E. coli BL21 (DE3)/pET32a (+)-Glu was inoculated in 5 mL LB liquid medium (containing 100 μg/mL ampicillin) and cultured at 37°C overnight. Then, 1 mL of this culture was inoculated into 100 mL LB medium (containing 100 μg/mL ampicillin) and grown at 37°C with shaking at 200 rpm until the OD600 value reached 0.4-0.6.

Publication protocol

The recombinant strain E. coli BL21 (DE3)/pET32a (+)-Glu was inoculated in 5 mL LB liquid medium (containing 100 μg/mL ampicillin) and cultured at 37°C overnight. Then, 1 mL of this culture was inoculated into 100 mL LB medium (containing 100 μg/mL ampicillin) and grown at 37°C with shaking at 200 rpm until the OD600 value reached 0.4-0.6. At this time, IPTG was added at a final concentration of 0.5, 1, 2, 4, 6, or 8 mM, and the culture was incubated for another 6 h at 30°C, shaking at 200 rpm. For the controls, E. coli BL21 (DE3)/pET32a strain induced with 1 mM IPTG and E. coli BL21 (DE3)/pET32a Glu without IPTG induction were used. The cell pellet from 1-mL culture, obtained by centrifugation at 10,621 g for 5 min, was resuspended in 100 μL 20 mM PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4, and 1.8 mM KH2PO4, pH 7.3) and 8μL lysozyme (50 mg/L) at 37°C for 1 h. The mixture was then subjected to 4-5 freeze-thaw cycles using liquid nitrogen and the supernatant was obtained by centrifugation at 15,294 g for 10 min. Finally, 15 μL of this supernatant was analyzed by sodium dodecyl sulfate (SDS)-PAGE using the Mini-protean III system (Bio-Rad, Beijing, China). Protein bands were visualized by Coomassie brilliant blue R-250 staining.

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