pET20b-chIL-7/H

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	Escherichia coli BL21 (DE3) competent cells were transformed with the pET20b-chIL-7/H vector, and positive transformed E. coli were cultured overnight in 5 mL Luria–Bertani (LB) medium containing 100 μg/mL ampicillin at 37 °C. When the optical density at 600 nm (OD600) of the culture reached 0.6, isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to induce chIL-7 expression.

Protocol tips

Escherichia coli BL21 (DE3) competent cells were transformed with the pET20b-chIL-7/H vector, and positive transformed E. coli were cultured overnight in 5 mL Luria–Bertani (LB) medium containing 100 μg/mL ampicillin at 37 °C. When the optical density at 600 nm (OD600) of the culture reached 0.6, isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to induce chIL-7 expression.

Publication protocol

Escherichia coli BL21 (DE3) competent cells were transformed with the pET20b-chIL-7/H vector, and positive transformed E. coli were cultured overnight in 5 mL Luria–Bertani (LB) medium containing 100 μg/mL ampicillin at 37 °C. Cultures were then added to fresh 100 mL LB media in 500 mL flasks and allowed to continue to grow at 30 °C with shaking (200 rpm). When the optical density at 600 nm (OD600) of the culture reached 0.6, isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to induce chIL-7 expression. The E. coli were cultured for four additional hours and then harvested by centrifugation at 8000 × g for 5 min at 4 °C. Bacterial pellets were resuspended in 30 mL suspension buffer (30 mM Tris–HCl, 20% sucrose, pH8) and 60 μL 0.5 mol/L EDTA (pH8) was added into the suspension (final concentration 1 mM) and stirred for 10 min at room temperature. The bacteria were collected by centrifugation at 4 °C for 10 min at 8000 g, resuspended in 30 mL ice-cold 5 mM MgSO4, and stirred in an ice bath for 10 min, resulting in the release of periplasmic proteins including chIL-7 into the medium. Following centrifugation at 10 000 g for 10 min at 4 °C, the supernatant containing recombinant chIL-7 was collected for purification.

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