期刊
INTERNATIONAL IMMUNOPHARMACOLOGY
卷 39, 期 -, 页码 181-191出版社
ELSEVIER
DOI: 10.1016/j.intimp.2016.07.029
关键词
Antibacterial peptide; Recombinant expression; Intein system; Enterotoxigenic Escherichia coli-K88; Lactoferricin P; Immunomodulatory
资金
- earmarked fund for Modern Agro-industry Technology Research System, China [CARS-36]
LF-6 is a modified antibacterial peptide derived from LFP-20, a major active ingredient of porcine lactoferrin, whose antibacterial activity is 200 times higher than its native protein counterpart. Moreover, LF-6 displays even higher antibacterial activity than LFP-20 and negligible toxic adverse effects, make it a potential therapeutic agent for antibacterial purposes. Escherichia coli expression system has been a preferred choice and workhorse for most recombinant proteins. However, LF-6 must be coexpressed with a fusion partner to avoid its potentially fatal toxicity which would threat E. coli expression system. In this study, we successfully introduced intein system to solve this problem, which LF-6 was N-terminally fused to dithiothreitol (DTT)-induced self-cleavable intein, and it conduct cleavage when the intein-fusion peptide passing through a chromatography column filled with chitin, then the spliced peptide was purified with RP-HPLC and identified with mass spectroscopy. A bacteriostatic test showed that the recombinant LF-6 displayed nearly the same antibacterial activity as the chemically synthetized LF-6, and an in vivo immunoprotection analysis showed that the recombinant LF-6 exerted protective effects on Escherichia coli (ETEC)-K88-infected mice, which significantly reduced the pro-inflammatory cytokines level in plasma and intestine, and resistant to intestinal mucosal injury compared to the infective alone groups. Our study indicates that the intein system allows a safe and efficient method to produce recombinant LF-6, which not only has antibacterial activity, but more importantly, has an immunomodulatory function. (C) 2016 Elsevier B.V. All rights reserved.
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