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Construction and expression of rabbit neutrophil peptide-1 gene in Escherichia coli

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Abstract

Rabbit neutrophil peptide-1 (NP-1) is a prototypic rabbit α-defensin with a broad antimicrobial spectrum. The coding sequence of NP-1 was amplified and cloned into pET-31b(+) to construct an expression vector, pET31-NP1, which was transformed into E. coli BLR(DE3)pLysS for expressing the fusion NP-1 protein (fNP-1). The fNP-1 is downstream of a ketosteroid isomerase (KSI) and upstream of a (His)6-Tag, as KSI-NP1-His6. The optimal condition, cultivation in enriched LB medium and induction with 0.5 mM IPTG for 6 h, was determined for fNP-1 production. The fNP-1 was purified by Ni-NTA resin and cleaved by cyanogen bromide to release matured NP-1 peptide. The matured NP-1 peptide showed significant antimicrobial activities against clinical bacteria, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. The application of this expression approach represents a potential method to produce NP-1 by fusion protein expressed in E. coli without cytotoxicity.

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Acknowledgements

The authors gratefully acknowledge Dr. Shih-Rong Wang at the Animal Technology Institute Taiwan for the gift of rat anti-NP1 antiserum. This work was supported by the grant of 96-EC-17-A-17-R7-0454 from the Ministry of Economic Affairs of Executive Yuan, Taiwan.

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Authors and Affiliations

  1. Division of Biotechnology, Animal Technology Institute Taiwan, P.O. Box 23, Chunan, 35099, Miaoli, Taiwan

    Yu Ling Sun, Yi Juain Lin & Yu Chi Chou

  2. Department of Biological Science and Technology, National Chiao Tung University, No.75 Po-Ai Street, Hsinchu, 30068, Taiwan

    Tang Ching Kuan & Chih Sheng Lin

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  1. Yu Ling Sun
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  2. Tang Ching Kuan
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  3. Yi Juain Lin
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Correspondence to Chih Sheng Lin.

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Sun, Y.L., Kuan, T.C., Lin, Y.J. et al. Construction and expression of rabbit neutrophil peptide-1 gene in Escherichia coli . Ann Microbiol 60, 329–334 (2010). https://doi.org/10.1007/s13213-010-0046-z

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  • DOI: https://doi.org/10.1007/s13213-010-0046-z

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