title: Recombinant expression of porcine lactoferrin peptide LF-6 with intein technology and its immunomodulatory function in ETEC K88-infected mice.
abstract: 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.
authors.affiliation: Institute of Feed Science, Zhejiang University, Key Laboratory of Animal Nutrition and Feed Science, Ministry of Agriculture (East China), Zhejiang Provincial Laboratory of Feed and Animal Nutrition, Hangzhou, Zhejiang 310058, People's Republic of China. Electronic address: 84413414@qq.com.
authors.forename: Qin
authors.initials: Q
authors.lastname: Jiang
authors.affiliation: Laboratory of Tropical Animal Breeding, Reproduction and Nutrition, Hainan University, Haikou, Hainan 570228, People's Republic of China. Electronic address: 007zhanghaiwen@163.com.
authors.forename: Haiwen
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Institute of Feed Science, Zhejiang University, Key Laboratory of Animal Nutrition and Feed Science, Ministry of Agriculture (East China), Zhejiang Provincial Laboratory of Feed and Animal Nutrition, Hangzhou, Zhejiang 310058, People's Republic of China. Electronic address: xyg285@gmail.com.
authors.forename: Yonggang
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Institute of Feed Science, Zhejiang University, Key Laboratory of Animal Nutrition and Feed Science, Ministry of Agriculture (East China), Zhejiang Provincial Laboratory of Feed and Animal Nutrition, Hangzhou, Zhejiang 310058, People's Republic of China. Electronic address: zhanghwas@163.com.
authors.forename: Yizhen
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000627:therapeutic use*; D016207:Cytokines / Q000097:blood; D054307:Enterotoxigenic Escherichia coli / Q000276:immunology*; D004927:Escherichia coli Infections / Q000188:drug therapy*; D005240:Feasibility Studies; D007155:Immunologic Factors / Q000627:therapeutic use*; D018836:Inflammation Mediators / Q000097:blood; D047668:Inteins / Q000235:genetics; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000276:immunology / Q000382:microbiology; D007781:Lactoferrin / Q000235:genetics / Q000627:therapeutic use*; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D010446:Peptide Fragments / Q000235:genetics / Q000627:therapeutic use*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013552:Swine
chemical_list: D000890:Anti-Infective Agents; D016207:Cytokines; D007155:Immunologic Factors; D018836:Inflammation Mediators; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D007781:Lactoferrin
keywords: Antibacterial peptide; Enterotoxigenic Escherichia coli-K88; Immunomodulatory; Intein system; Lactoferricin P; Recombinant expression