Antibacterial peptide Hex-Mag

According to the partiality codon of Pichia pastoris, hybrid antimicrobial peptide CecA-mag gene was synthesized and cloned into pPICZa-A to construct the recombinant expression vector pPICZa-A-CA. The SacI-linearized plasmid pPICZalpha-A-CA was transformed into P. pastoris SMD1168 by electroporation. Under the control of the promoter AOX'(alcoholoxidase') , an approximately 1.9kDa cecA-mag protein was expressed. Antibacterial assays demonstrated that cecA-mag had broad spectrum of antimicrobial property against Gram-positive as well as Gram-negative bacteria especially showed potent antibacterial activity against ampicillin resistant bacteria, such as pathogenic E. coli. In addition, the hybrid antibacterial peptide showed an extreme heat stable and acid stable characteristic. These results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional cecA-mag for both research and industrial purpose. Based on these characteristics, the recombinant antibacterial peptide cecA-mag displays application foreground in the field of prevention of disease, and can be used as additives of animal feedstuff and so on.

The hybrid peptide CA(1-7)-M(2-12) gene was designed according to the N-terminal 1-7 amino acid sequence of the antimicrobial peptide cecropin A (CA) and the N-terminal 2-12 amino acid sequence of maganin (M) and synthesized using Pichia pastoris preferred codons. The gene was cloned into pPICZαA and transformed into the P. pastoris recipient bacterium SMD1168, regulated by the alcohol oxidase (AOX). Expression of the cecA-mag hybrid antimicrobial peptide (MW, 1.9 kDa) revealed broad-spectrum antibiotic activity and to the ability to inhibit growth of most G(-) and G(+) bacteria. Three mutants of cecA-mag were designed and synthesized by recombination polymerase chain reaction site-directed mutagenesis to investigate the relationship between the structure and function of this antimicrobial peptide. The inhibition titers of these mutants against Staphylococcus aureus were evaluated using the agar diffusion method. Under the conditions of the same concentration and volume, the bacteriostatic diameters of three cecA-mag mutants were 1.2, 1.2 and 1.5 times, respectively, compared with the diameters of wild-type cecA-mag.

According to the partiality codon of Pichia pastoris, hybrid antimicrobial peptide CecA-mil gene was reconstructed, synthesized and cloned into pPICZalpha-A to construct the recombinant expression vector pPICZa-A-CM. The pPICZalpha-A-CM was transformed into yeast host strain X-33. Under the control of the promoter AOX1 (alcohol oxidase 1), a approximately 1.9 kD cecA-mil protein was expressed with the high level of 245 microg/mL after optimized the requirements for the flask-shaking culture fermentation of the Pichia pastoris rX-33/pPICZalpha-A-CM. The hybrid antibacterial peptide had a broad spectrum antibacterial activity on both gram-positive and gram-negative bacteria, especially showed potent antibacterial activity against ampicillin-resistant and kanamycin- resistant bacteria, such as Staphylococcus aureus (cowan I) and pathogenic E. coli (O1) from chicken. In addition, the hybrid antibacterial peptide showed an extreme heat-stable and acid-stable characteristic. Based on these characteristics, the recombinant antibacterial peptide CecA-mil display application foreground in the field of antisepsis of food, prevention of disease, additives of animal feedstuff and so on.