pET28a::BPL3

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	The transformants were inoculated in LB medium containing kanamycin (25 μg/mL) and cultured overnight at 37 °C. The subculture was diluted 1:100 in 100 mL of LB medium and grown for 1–4 h at 37 °C in an Erlenmeyer flask in a shaker until reaching OD 0.4–0.6. When the designated OD was reached, 1 mL of sample was removed from the flask and the cell pellet was collected by centrifugation (un-induced control). To induce the protein, IPTG (final concentration, 0.4 mM) was added and cultured at various temperatures (20 °C, 25 °C, 30 °C, and 37 °C) overnight.

Protocol tips

The transformants were inoculated in LB medium containing kanamycin (25 μg/mL) and cultured overnight at 37 °C. The subculture was diluted 1:100 in 100 mL of LB medium and grown for 1–4 h at 37 °C in an Erlenmeyer flask in a shaker until reaching OD 0.4–0.6. When the designated OD was reached, 1 mL of sample was removed from the flask and the cell pellet was collected by centrifugation (un-induced control). To induce the protein, IPTG (final concentration, 0.4 mM) was added and cultured at various temperatures (20 °C, 25 °C, 30 °C, and 37 °C) overnight.

Publication protocol

"The transformants were inoculated in LB medium containing kanamycin (25 μg/mL) and cultured overnight at 37 °C. The subculture was diluted 1:100 in 100 mL of LB medium and grown for 1–4 h at 37 °C in an Erlenmeyer flask in a shaker until reaching OD 0.4–0.6. When the designated OD was reached, 1 mL of sample was removed from the flask and the cell pellet was collected by centrifugation (un-induced control). To induce the protein, IPTG (final concentration, 0.4 mM) was added and cultured at various temperatures (20 °C, 25 °C, 30 °C, and 37 °C) overnight. To determine the optimum concentration of IPTG, various concentrations of IPTG (0.1, 0.2, 0.4, and 1 mM) were added to the bacterial culture (OD 0.4–0.6) overnight. The effects of various incubation times (1, 3, and 5 h and overnight) were determined by incubating bacteria after the addition of IPTG (0.4 mM). Aliquots (5 mL) were collected after incubation in different conditions.

Total proteins from each cell culture were extracted and analyzed to determine the expression levels of rD1BPL3 and rD2BPL3. Total protein extracts were prepared by heating samples for 5 min at 90 °C after treatment in 1× SDS-PAGE sample buffer (0.2 mL/mL culture) to the cell precipitates. The extracts were centrifuged at 20,000× g and the supernatants were used directly for SDS-PAGE. Soluble fractions were obtained from cell precipitates after cell culture (5 mL). Resuspended samples in 1 mL of extraction buffer (1× PBS, 10 mM imidazole, 1 mM PMSF, pH 7.2) were sonicated at a 15% amplitude, repeated 20 times, with 3-second on/off periods. Supernatants were collected by centrifugation at 20,000× g for 10 min. The expression efficiency was determined by calculating the target band intensity after SDS-PAGE using GelAnalyzer 2010 (http://www.gelanalyzer.com/). 4.4. Purification of rBPL3."

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