pG-COE

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	A seed culture was prepared by inoculating with a single colony of recombinant E.coli strains and grown overnight in 5 ml of LB broth containing 100 μg/ml of ampicillin in a 50 ml sterile plastic tubes. For the expression of recombinant proteins, 1 % seed culture was routinely inoculated to 25 ml of LB broth containing 0.5 mg/ml of L-arabinose, 100 μg/ml of ampicillin and 20 μg/ml of chloramphenicol in 100 ml baffled flasks, followed by adding L-arabinose (0.5 mg/ml) and/or tetracycline (10 ng/ml) for the induction of chaperone proteins. Cells were cultured with shaking at 230 rpm and 37 °C. The growth was monitored by measuring the absorbance of optical density (OD) at 600 nm with a spectrophotometer. When the OD600 of the culture reaches set point, cultures were cooled to 4 °C for 30 min and induced by 0.1 mM IPTG at 15 °C for 24 h.

Protocol tips

A seed culture was prepared by inoculating with a single colony of recombinant E.coli strains and grown overnight in 5 ml of LB broth containing 100 μg/ml of ampicillin in a 50 ml sterile plastic tubes. For the expression of recombinant proteins, 1 % seed culture was routinely inoculated to 25 ml of LB broth containing 0.5 mg/ml of L-arabinose, 100 μg/ml of ampicillin and 20 μg/ml of chloramphenicol in 100 ml baffled flasks, followed by adding L-arabinose (0.5 mg/ml) and/or tetracycline (10 ng/ml) for the induction of chaperone proteins. Cells were cultured with shaking at 230 rpm and 37 °C. The growth was monitored by measuring the absorbance of optical density (OD) at 600 nm with a spectrophotometer. When the OD600 of the culture reaches set point, cultures were cooled to 4 °C for 30 min and induced by 0.1 mM IPTG at 15 °C for 24 h.

Publication protocol

"A seed culture was prepared by inoculating with a single colony of recombinant E.coli strains and grown overnight in 5 ml of LB broth containing 100 μg/ml of ampicillin in a 50 ml sterile plastic tubes. For the expression of recombinant proteins, 1 % seed culture was routinely inoculated to 25 ml of LB broth containing 0.5 mg/ml of L-arabinose, 100 μg/ml of ampicillin and 20 μg/ml of chloramphenicol in 100 ml baffled flasks, followed by adding L-arabinose (0.5 mg/ml) and/or tetracycline (10 ng/ml) for the induction of chaperone proteins. Cells were cultured with shaking at 230 rpm and 37 °C. The growth was monitored by measuring the absorbance of optical density (OD) at 600 nm with a spectrophotometer. When the OD600 of the culture reaches set point, cultures were cooled to 4 °C for 30 min and induced by 0.1 mM IPTG at 15 °C for 24 h.

To check the chaperone effect of trigger factor, E.coli BL21/pTf16/pG-COE and E.coli BL21/pTf16/pG-S1D were shake-cultured in LB broth at 37 °C until OD600 reached 0.6, cooled to 4 °C for 30 min and induced at 15 °C for 24 h with or without IPTG (0.1 mM) and with or without L-arabinose (0.5 mg/ml) to detect the protein expression.

Cells were harvested and disrupted on ice by sonication (VCX 750, SONICS, Newtown, CT, USA) using a program (45 cycles of 2 sec On/5 s Off, amp 20 %) and centrifuged at 20,000 g for 15 min at 4 °C. The pellets and supernatants were separately stored at −20 °C until use. Protein concentration was measured by Bradford assay (BioRad, CA, USA) using bovine serum albumin (BSA) as a standard. Protein expression was evaluated using band intensities of recombinant protein on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) from independent and/or parallel induction samples."

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