pHT43-BMP2-M

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	A single positive transformant of each pHT43-BMP2-M and pHT43-BMP2-D plasmid was inoculated into 5 ml LB medium (5 g/L yeast extract, 10 g/L tryptone and 10 g/L NaCl) containing 5 μg/ml chloramphenicol and incubated at 37°C, 200 rpm for overnight. Next morning, 25 ml LB medium was refreshed with 2% overnight culture and incubated under the conditions mentioned above until the OD600 reached 0.6–0.8. IPTG (1 mM) was induced to obtain the expression of the rhBMP2 monomer and dimer. A postinduction sample was taken at 2, 4, and 6 hours and cells were harvested by centrifugation at 13000 rpm for 5 min at 4°C.	The culture supernatant was processed further for the confirmation of secretory protein expression with trichloroacetic acid (TCA) precipitation.

Protocol tips
A single positive transformant of each pHT43-BMP2-M and pHT43-BMP2-D plasmid was inoculated into 5 ml LB medium (5 g/L yeast extract, 10 g/L tryptone and 10 g/L NaCl) containing 5 μg/ml chloramphenicol and incubated at 37°C, 200 rpm for overnight. Next morning, 25 ml LB medium was refreshed with 2% overnight culture and incubated under the conditions mentioned above until the OD600 reached 0.6–0.8. IPTG (1 mM) was induced to obtain the expression of the rhBMP2 monomer and dimer. A postinduction sample was taken at 2, 4, and 6 hours and cells were harvested by centrifugation at 13000 rpm for 5 min at 4°C.
Downstream tips
The culture supernatant was processed further for the confirmation of secretory protein expression with trichloroacetic acid (TCA) precipitation.

Publication protocol

A single positive transformant of each pHT43-BMP2-M and pHT43-BMP2-D plasmid was inoculated into 5 ml LB medium (5 g/L yeast extract, 10 g/L tryptone and 10 g/L NaCl) containing 5 μg/ml chloramphenicol and incubated at 37°C, 200 rpm for overnight. Next morning, 25 ml LB medium was refreshed with 2% overnight culture and incubated under the conditions mentioned above until the OD600 reached 0.6–0.8. IPTG (1 mM) was induced to obtain the expression of the rhBMP2 monomer and dimer. A postinduction sample was taken at 2, 4, and 6 hours and cells were harvested by centrifugation at 13000 rpm for 5 min at 4°C. The culture supernatant was processed further for the confirmation of secretory protein expression with trichloroacetic acid (TCA) precipitation. Briefly 0.15% sodium deoxycholate was added in the culture medium and incubated at room temperature for 15 min. After that 150 μl of 100% TCA solution was added and incubated for 10 min on ice. The sample was then centrifuged at 14000 rpm at 4°C for 10 min. The supernatant was discarded and the pellet was washed with 1 ml of 100% acetone solution twice and centrifuged at 14000 rpm for 10 min at 4°C during washing. The pellet was resuspended in 100 μl of Tris-Cl buffer (pH 8.0) and run on 18% SDS-PAGE for expression analysis. After confirmation, the maximum expression of rhBMP2 monomer protein was optimized in three different media [LB, 2xYT (16 g/L tryptone, 5 g/L NaCl, 10 g/L yeast extract) and 2xL MAL (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose hydrate, and 7.5 μg/mL MnSO4)] and at two different temperatures, that is, 30°C and 37°C in SCK6 and WB600 strains. The optimization of rhBMP2 monomer was done initially and then the dimer was grown on the preoptimized conditions of media, temperature, and the strain. Though, monomer and dimer were optimized individually in case of time span, IPTG, and lactose concentration.

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