Browsing by Author "Lin, Yi-Juain"

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Soluble Fusion Expression and Production of Rabbit Neutrophil Peptide-1 in Escherichia coli
(2011-09-01)
Sun, Yu-Ling
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Lin, Yi-Juain
;
Lin, Chih-Sheng
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生物科技學系
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Department of Biological Science and Technology
Rabbit neutrophil peptide-1 (NP1) is a prototypic a-defensin with a broad antimicrobial spectrum. The Escherichia coli-preferential coding sequence of rabbit mature NP-1 (maNP1) was designed, amplified and cloned into plasmid pET32b(+) to construct an expression vector. pET32-maNP1. This expression vector was transformed into E. coli Rosetta-gami(DE3)pLysS for expressing the fusion protein, Trx-(His)(6)-maNP1, i.e., expressed maNP1 is sequentially downstream of a thioredoxin (Trx) and a (His)(6)-tag. The quantitative data showed that there were 52% and 4.5% of the fused Trx-(His)(6)-maNP1 expressed in the soluble and insoluble form in the E. cola carrying pET32-maNP1 with IPTG induction, respectively. The produced fusion protein was collected and purified by Ni-NTA resin, and then cleaved by enterokinsase to release maNP1 peptide. The purified recombinant maNP1 showed significantly antimicrobial activities against clinical bacteria, E. coli and Pseudomonas aeruginosa (Gram-negative) and Staphylococcus aureus and Bacillus subtilis (Gram-positive). The application of this expression approach represents a potential method to produce functional maNP1 by fusion protein expressed in E. coli.