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. 2014 Jul-Aug;61(4):466-73.
doi: 10.1002/bab.1194. Epub 2014 Mar 25.

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

Jianfeng Wang  1   2 , Zhiqiang Xiong  1 , Yingying Yang  1   2 , Na Zhao  1 , Yong Wang  1
Affiliations
Free PMC article

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

Jianfeng Wang et al. Biotechnol Appl Biochem. 2014 Jul-Aug.
Free PMC article

Abstract

Heterologous expression is an efficient alternative to conventional extraction to produce a specific Buthus martensii Karsch (BmK) peptide. In this work, BmK1 was successfully expressed in Escherichia coli after genetic codon optimization, but BmK1 content was <6% of total cellular protein. To improve BmK1 expression, a trc promoter library with a wide relative strength was constructed, and three promoters, PpJF136 (0.55), PpJF325 (1.29), and PpJF288 (2.31), were selected to control BmK1 expression. A higher BmK1 expression (>13.9% of total protein) was obtained using a stronger promoter, PpJF325 . Furthermore, a maximum BmK1 content (>21.7% of total protein) was obtained by combining promoter PpJF325 and three copies of the BmK1 gene. 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.

Keywords: BmK1; copy number; heterologous expression; promoter engineering; scorpion venom.

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Figures

Figure 1
Figure 1
Codon optimization and expression cassette construction. (A) DNA sequence alignment between original and optimized BmK1 gene. (B) The entire open reading frame of the recombinant BmK1 gene.
Figure 2
Figure 2
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.
Figure 3
Figure 3
Precise characterization of trc promoter library.
Figure 4
Figure 4
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.
Figure 5
Figure 5
Effect of gene dosage on BmK1 expression. (A) Multicopy expression cassette construction. (B) Effect of gene dosage on cell growth. (C) Effect of gene dosage on BmK1 yield.
Figure 6
Figure 6
Quantification of soluble BmK1 peptide in culture. (A) SDS-PAGE analysis of the purified BmK1 peptide. (B) The yield of the purified BmK1 peptide.
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