title: Cloning, expression and characterization of antimicrobial porcine β defensin 1 in Escherichia coli.
abstract: Porcine β defensin 1 (pBD1) is a cationic antimicrobial peptide with three pairs of disulfide bonds. When expressed in insect cells, two polypeptides of different length (pBD1(38) and pBD1(42)) accumulated, which differed by N-terminal truncation. However, only pBD1(42) was found in pigs. pBD1(42) had stronger antimicrobial activity than pBD1(38), and thus could be a good candidate as a bactericidal agent for pigs. In this study, pBD1(42) gene, obtained by RT-PCR using the tongue total RNA as a template, was cloned into pET30a expression vector and transformed into Escherichia coli BL21 (DE3) plysS. The recombinant pBD1(42) was expressed after induction by IPTG and purified by His tag affinity column with 90% purity. The recombinant pBD1(42) exhibited antimicrobial activity against both Gram-positive Staphylococcus aureus and Gram-negative E. coli including the multi-resistant E. coli. The minimum inhibitory concentrations (MICs) of recombinant pBD1(42) against tested bacteria were 100 μg/mL for E. coli and 80 μg/mL for S. aureus. In addition, pBD1(42) showed low hemolytic activity and high thermal stability. These properties are relevant for the biotechnological applications of the peptide.
authors.affiliation: Department of Animal and Veterinary Science, Henan Agricultural University, Zhengzhou 450002, Henan, The People's Republic of China. hnclli@163.com
authors.forename: Chun-li
authors.initials: CL
authors.lastname: Li
authors.forename: Ting-ting
authors.initials: TT
authors.lastname: Xu
authors.forename: Rui-bo
authors.initials: RB
authors.lastname: Chen
authors.forename: Xian-xian
authors.initials: XX
authors.lastname: Huang
authors.forename: Yan-cong
authors.initials: YC
authors.lastname: Zhao
authors.forename: Yuan-yuan
authors.initials: YY
authors.lastname: Bao
authors.forename: Wei-dong
authors.initials: WD
authors.lastname: Zhao
authors.forename: Zhen-yu
authors.initials: ZY
authors.lastname: Zheng
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000302:isolation & purification; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D003001:Cloning, Molecular; D018432:Drug Resistance, Multiple / Q000187:drug effects / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects; D015870:Gene Expression; D008826:Microbial Sensitivity Tests; D013211:Staphylococcus aureus / Q000187:drug effects; D013552:Swine; D023083:beta-Defensins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D023083:beta-Defensins