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

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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; Cys residues; antimicrobial peptide; disulfide bonds; expression; hemolytic activity; pichia pastoris; recombinant plasmids

Document Type: Research Article

DOI: http://dx.doi.org/10.2174/092986612802762660

Publication date: October 1, 2012

More about this publication?
  • Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.
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