title: Design and high-level expression of a hybrid antimicrobial peptide LF15-CA8 in Escherichia coli.
abstract: Antimicrobial peptides (AMPs) have been paid considerable attention owing to their broad-spectrum antimicrobial activity and have great potential as novel antimicrobials. In this study, a novel hybrid peptide LF15-CA8 was designed on the basis of bovine lactoferricin (LfcinB) and cecropin A. The gene segment encoding LF15-CA8 was synthesized and cloned into pGEX-4T-BH to form pGEX-4T-LC1 containing one copy of the LF15-CA8 coding region. A series of recombinant vectors containing up to six multiple-copy LF15-CA8 coding regions, i.e., pGEX-4T-LCn (n = 1-6), were subsequently constructed, and used for transformation in Escherichia coli BL21(DE3). After induction with IPTG, pGEX-4T-LC1 and pGEX-4T-LC2 transformants successfully expressed fusion proteins GST-LF15-CA8 and GST-(LF15-CA8)₂ in the form of inclusion bodies, respectively. The inclusion bodies were dissolved and the peptide was successfully released in 70 % formic acid in a single step. After purification, about 10.0 mg of the recombinant peptide LF15-CA8 with purity more than 97 % was obtained from 1 l of bacteria culture of pGEX-4T-LC2 transformants. LF15-CA8 caused an increase in antibacterial activity against Gram-positive bacterium (Staphylococcus aureus ATCC 25923) compared with the parent peptides and did not show obvious hemolytic activity against human erythrocytes in the range of effective antibacterial concentration. These results suggest that the peptide LF15-CA8 could be a promising candidate for therapeutic applications, and may lead to a cost-effective solution for the large-scale production of AMPs.
authors.affiliation: College of Animal Science and Technology, Northeast Agricultural University, 59 Mucai Street, Xiangfang District, Harbin, 150030, China, fengxingjun2008@163.com.
authors.forename: Xing-Jun
authors.initials: XJ
authors.lastname: Feng
authors.forename: Li-Wei
authors.initials: LW
authors.lastname: Xing
authors.forename: Di
authors.initials: D
authors.lastname: Liu
authors.forename: Xue-Ying
authors.initials: XY
authors.lastname: Song
authors.forename: Chun-Long
authors.initials: CL
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Wen-Shan
authors.initials: WS
authors.lastname: Xu
authors.forename: Zhong-Qiu
authors.initials: ZQ
authors.lastname: Li
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007781:Lactoferrin / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000187:drug effects
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D011993:Recombinant Fusion Proteins; C082171:lactoferricin B; C031161:cecropin A; D007781:Lactoferrin