title: Expression in Pichia Pastoris and biological activity of Avian β-defensin 6 and its mutant peptide without cysteines.
abstract: Avian β-defensin 6 (AvBD-6) is an antimicrobial peptide that plays significant roles in the innate immunity of chickens. To explore the effects of disulfide bonds on antimicrobial activity of AvBD-6, two peptides with or without Cys residues were designed and expressed in Pichia Pastoris. The peptide AvBD-6-B was obtained by removing six Cys residues of AvBD-6. According to the codon bias of Pichia Pastoris, the genes of AvBD-6 and AvBD-6-B were synthesized. The Bgl II-linearized recombinant plasmids pGAPHαM-AvBD-6 and pGAPHαM-AvBD-6-B were transformed into Pichia Pastoris GS115 by electroporation. The recombinant AvBD-6 and AvBD-6-B were expressed in YPD for 48 h, 72 h and 96 h at 30 �C Tricine-SDS-PAGE analysis demonstrated that both AvBD-6 and AvBD-6-B were expressed in Pichia Pastoris. The concentration of recombinant AvBD-6 and AvBD-6-B reached 114.9 mg/l and 93.8 mg/l, respectively. The expression products exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria. Antimicrobial activity of AvBD-6-B suggests that the removal of six Cys residues had no significant effect on the antimicrobial activity of avian β-defensins. Neither peptide showed hemolytic activity. This study could serve as an impetus for the production of this antimicrobial peptide as a replacement for antibiotics in animal feed.
authors.affiliation: Laboratory of Molecular Nutrition and Immunity, Institute of Animal Nutrition, Northeast Agricultural University, Harbin, 150030, PR China.
authors.forename: Yanping
authors.initials: Y
authors.lastname: Cao
authors.forename: Qingquan
authors.initials: Q
authors.lastname: Ma
authors.forename: Anshan
authors.initials: A
authors.lastname: Shan
authors.forename: Na
authors.initials: N
authors.lastname: Dong
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology*; D030161:Avian Proteins / Q000235:genetics / Q000494:pharmacology*; D001483:Base Sequence; D002645:Chickens; D003545:Cysteine / Q000235:genetics*; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics / Q000494:pharmacology*; D010843:Pichia / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D023083:beta-Defensins / Q000235:genetics / Q000494:pharmacology*
chemical_list: D000890:Anti-Infective Agents; D030161:Avian Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins; D003545:Cysteine