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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.

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Effect of promoter strength on BmK1 expression. (A) Expression cassette construction using four different promoters. (B) Effect of promoter strength on cell growth. (C) Effect of promoter strength on BmK1 yield.
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fig04: Effect of promoter strength on BmK1 expression. (A) Expression cassette construction using four different promoters. (B) Effect of promoter strength on cell growth. (C) Effect of promoter strength on BmK1 yield.

Mentions: There was a low level of BmK1 expression when a single copy of the BmK1 gene was under the control of a wild-type trc promoter. We thus made an attempt to enhance BmK1 expression through promoter engineering. A single copy of the BmK1 gene was constructed under the control of four different promoters with a relative strength of 0.55 (PpJF136), 1.0 (Ptrc), 1.29 (PpJF325), and 2.31 (PpJF288), respectively, to evaluate the effect of promoter strength on the BmK1 expression (Fig.4A). An interesting phenomenon was found that cell growth was deteriorated accompanied with upregulation of promoter strength (Fig.4B). The growth of E. coli BL21 (DE3) pJF396 was almost completely stopped after 1 H of a prompt initiation of BmK1 expression. As shown in Fig.4C, BmK1 expression was initiated immediately after IPTG addition. When only induced for 0.5 H, BmK1 content can reach 7.37% and 9.11% in E. coli BL21 (DE3) pJF393 (PpJF325, strong promoter) and pJF396 (PpJF288, strong promoter), which were higher than those of pJF391 (Ptrc, control) and pJF392 (PpJF136, weak promoter). As for pJF396 with promoter strength at 2.31, BmK1 content was significantly lower than that of pJF393; it slightly decreased at 1 H after induction, which might be attributed to the inhibition of cell growth caused by a higher BmK1 expression/BmK1 toxicity. During the whole process, all cultures reached their maximum BmK1 production less than 4 H after induction. A strong promoter can rapidly start BmK1 expression with a higher rate and relatively decrease peptide degradation, resulting in a higher BmK1 accumulation in the cell. The maximum BmK1 content of 13.93% was obtained at 2 H after induction using pJF393, which was improved 2.27-fold compared with that of the control (6.12% of BmK1 content in pJF391).


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)

Effect of promoter strength on BmK1 expression. (A) Expression cassette construction using four different promoters. (B) Effect of promoter strength on cell growth. (C) Effect of promoter strength on BmK1 yield.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig04: Effect of promoter strength on BmK1 expression. (A) Expression cassette construction using four different promoters. (B) Effect of promoter strength on cell growth. (C) Effect of promoter strength on BmK1 yield.
Mentions: There was a low level of BmK1 expression when a single copy of the BmK1 gene was under the control of a wild-type trc promoter. We thus made an attempt to enhance BmK1 expression through promoter engineering. A single copy of the BmK1 gene was constructed under the control of four different promoters with a relative strength of 0.55 (PpJF136), 1.0 (Ptrc), 1.29 (PpJF325), and 2.31 (PpJF288), respectively, to evaluate the effect of promoter strength on the BmK1 expression (Fig.4A). An interesting phenomenon was found that cell growth was deteriorated accompanied with upregulation of promoter strength (Fig.4B). The growth of E. coli BL21 (DE3) pJF396 was almost completely stopped after 1 H of a prompt initiation of BmK1 expression. As shown in Fig.4C, BmK1 expression was initiated immediately after IPTG addition. When only induced for 0.5 H, BmK1 content can reach 7.37% and 9.11% in E. coli BL21 (DE3) pJF393 (PpJF325, strong promoter) and pJF396 (PpJF288, strong promoter), which were higher than those of pJF391 (Ptrc, control) and pJF392 (PpJF136, weak promoter). As for pJF396 with promoter strength at 2.31, BmK1 content was significantly lower than that of pJF393; it slightly decreased at 1 H after induction, which might be attributed to the inhibition of cell growth caused by a higher BmK1 expression/BmK1 toxicity. During the whole process, all cultures reached their maximum BmK1 production less than 4 H after induction. A strong promoter can rapidly start BmK1 expression with a higher rate and relatively decrease peptide degradation, resulting in a higher BmK1 accumulation in the cell. The maximum BmK1 content of 13.93% was obtained at 2 H after induction using pJF393, which was improved 2.27-fold compared with that of the control (6.12% of BmK1 content in pJF391).

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