Browsing by Author "Sun, Yu Ling"

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Construction and expression of rabbit neutrophil peptide-1 gene in Escherichia coli
(2010-06-01)
Sun, Yu Ling
;
Kuan, Tang Ching
;
Lin, Yi Juain
;
Chou, Yu Chi
;
Lin, Chih Sheng
Rabbit neutrophil peptide-1 (NP-1) is a prototypic rabbit alpha-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-His(6). 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.