Expression in Pichia pastoris and Biological Activity of Avian β-defensin 6 and its Mutant Peptide Without Cysteines

Yanping      Cao; Qingquan      Ma; Anshan      Shan; Na      Dong

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.

Keywords: Avian β-Defensin 6, disulfide bonds, expression, pichia pastoris, antimicrobial peptide, Cys residues, recombinant plasmids, hemolytic activity