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Significant expression of a Chinese scorpion peptide, BmK1, in Escherichia coli through promoter engineering and gene dosage strategy.

Wang J, Xiong Z, Yang Y, Zhao N, Wang Y - Biotechnol. Appl. Biochem. (2013)

Bottom Line: Heterologous expression is an efficient alternative to conventional extraction to produce a specific Buthus martensii Karsch (BmK) peptide.The yield of the purified BmK1 achieved 196.74 mg L(-1) in E. coli BL21(DE3) pJF431, which was improved 2.09-fold compared with the control.This was the highest reported production of scorpion peptides in E. coli.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People's Republic of China; State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People's Republic of China.

No MeSH data available.


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Expression of BmK1 using commercially available trc promoter. (A) Effect of temperature on BmK1 expression. Lane C, control strain E. coli BL21 (DE3) pTrcHis2B at 37 °C; lanes 1–3, BmK1 expression at 22 , 30 , and 37 °C, respectively. (B) Effect of IPTG addition on BmK1 expression. Lane C, control strain; lanes 1–4, expression at 0.1, 0.5, 1.0, and 2.0 mM of IPTG, respectively.
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fig02: Expression of BmK1 using commercially available trc promoter. (A) Effect of temperature on BmK1 expression. Lane C, control strain E. coli BL21 (DE3) pTrcHis2B at 37 °C; lanes 1–3, BmK1 expression at 22 , 30 , and 37 °C, respectively. (B) Effect of IPTG addition on BmK1 expression. Lane C, control strain; lanes 1–4, expression at 0.1, 0.5, 1.0, and 2.0 mM of IPTG, respectively.

Mentions: To achieve efficient expression of BmK1, a commercially available strong promoter trc was initially used to control the expression of a single-copy BmK1 gene. After 6 H of induction with 0.1 mM IPTG, BmK1 was successfully expressed, accounting for 4%–6% of total protein, with varied culture temperatures of 22, 30, and 37 °C, respectively (Fig.2A). It is indicated that the temperature does not significantly affect BmK1 expression, but the biomass of cultivation at 37 °C was much greater than those at the lower temperatures. We further investigated the effect of IPTG concentration at 37 °C. As for BmK1 expression, induction with 0.5 or 0.1 mM IPTG was obviously better than higher IPTG concentration (Fig.2B). Considering that cell growth of 0.1 mM IPTG was slightly better than 0.5 mM IPTG, the following study for BmK1 expression was carried out at 37 °C with 0.1 mM IPTG. The content of BmK1 reached 6.12% after 6 H induction using 0.1 mM IPTG at 37 °C; and cell growth slightly lagged behind the control (BL21[DE3] pTrcHis2B).


Significant expression of a Chinese scorpion peptide, BmK1, in Escherichia coli through promoter engineering and gene dosage strategy.

Wang J, Xiong Z, Yang Y, Zhao N, Wang Y - Biotechnol. Appl. Biochem. (2013)

Expression of BmK1 using commercially available trc promoter. (A) Effect of temperature on BmK1 expression. Lane C, control strain E. coli BL21 (DE3) pTrcHis2B at 37 °C; lanes 1–3, BmK1 expression at 22 , 30 , and 37 °C, respectively. (B) Effect of IPTG addition on BmK1 expression. Lane C, control strain; lanes 1–4, expression at 0.1, 0.5, 1.0, and 2.0 mM of IPTG, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4269186&req=5

fig02: Expression of BmK1 using commercially available trc promoter. (A) Effect of temperature on BmK1 expression. Lane C, control strain E. coli BL21 (DE3) pTrcHis2B at 37 °C; lanes 1–3, BmK1 expression at 22 , 30 , and 37 °C, respectively. (B) Effect of IPTG addition on BmK1 expression. Lane C, control strain; lanes 1–4, expression at 0.1, 0.5, 1.0, and 2.0 mM of IPTG, respectively.
Mentions: To achieve efficient expression of BmK1, a commercially available strong promoter trc was initially used to control the expression of a single-copy BmK1 gene. After 6 H of induction with 0.1 mM IPTG, BmK1 was successfully expressed, accounting for 4%–6% of total protein, with varied culture temperatures of 22, 30, and 37 °C, respectively (Fig.2A). It is indicated that the temperature does not significantly affect BmK1 expression, but the biomass of cultivation at 37 °C was much greater than those at the lower temperatures. We further investigated the effect of IPTG concentration at 37 °C. As for BmK1 expression, induction with 0.5 or 0.1 mM IPTG was obviously better than higher IPTG concentration (Fig.2B). Considering that cell growth of 0.1 mM IPTG was slightly better than 0.5 mM IPTG, the following study for BmK1 expression was carried out at 37 °C with 0.1 mM IPTG. The content of BmK1 reached 6.12% after 6 H induction using 0.1 mM IPTG at 37 °C; and cell growth slightly lagged behind the control (BL21[DE3] pTrcHis2B).

Bottom Line: Heterologous expression is an efficient alternative to conventional extraction to produce a specific Buthus martensii Karsch (BmK) peptide.The yield of the purified BmK1 achieved 196.74 mg L(-1) in E. coli BL21(DE3) pJF431, which was improved 2.09-fold compared with the control.This was the highest reported production of scorpion peptides in E. coli.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People's Republic of China; State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People's Republic of China.

No MeSH data available.


Related in: MedlinePlus