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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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Precise characterization of trc promoter library.
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fig03: Precise characterization of trc promoter library.

Mentions: To achieve an improved expression of BmK1 through promoter engineering, a promoter library of trc (an IPTG inducible strong promoter) was constructed and precisely characterized by detection of the relative strength. Considering that mRNA transcription and protein translation are two major genetic processes for gene expression, a fragment of 224 bp DNA sequence containing −10 and −35 core region of the trc promoter and RBS region (from 187 to 410 bp on pTrcHis2B) was subjected to random mutagenesis by error-prone PCR. Fifteen mutated promoters isolated from >1,000 mutants by primary screening were further precisely strength quantified at the induced and uninduced stages, ranging from 0.03 to 2.31 of relative strength after IPTG induction (Fig.3). Promoters PpJF136 (0.55), PpJF325 (1.29), and PpJF288 (2.31) with a low level of leaky expression were selected to fine-tune BmK1 expression in the following study. The sequences of three selected promoters are shown in Text S1 in the Supporting Information.


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)

Precise characterization of trc promoter library.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: Precise characterization of trc promoter library.
Mentions: To achieve an improved expression of BmK1 through promoter engineering, a promoter library of trc (an IPTG inducible strong promoter) was constructed and precisely characterized by detection of the relative strength. Considering that mRNA transcription and protein translation are two major genetic processes for gene expression, a fragment of 224 bp DNA sequence containing −10 and −35 core region of the trc promoter and RBS region (from 187 to 410 bp on pTrcHis2B) was subjected to random mutagenesis by error-prone PCR. Fifteen mutated promoters isolated from >1,000 mutants by primary screening were further precisely strength quantified at the induced and uninduced stages, ranging from 0.03 to 2.31 of relative strength after IPTG induction (Fig.3). Promoters PpJF136 (0.55), PpJF325 (1.29), and PpJF288 (2.31) with a low level of leaky expression were selected to fine-tune BmK1 expression in the following study. The sequences of three selected promoters are shown in Text S1 in the Supporting Information.

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