Expression and biological characterization of cecropin P1 from Ascaris in Pichia pastoris.

Objective To express the Ascaris antibacterial peptide cecropin P1 in Pichia pastoris and determine the activity of the expressed product.Methods Three oligonucleotide fragments were synthesized according to the codon bias of P.pastoris.The cecropin P1 gene was amplified by PCR and cloned into a pMD18-T vector,and a pMD18-T-CecP1 recombinant plasmid was constructed.After double digestion of pMD18-T-CecP1,the cecropin P1 gene was cloned into the vector pPICZα-A signal sequence of α-factor to construct the yeast secretory expression vector pPICZα-A-CecP1.The constructed recombinant plasmid pPICZα-A-CecP1 was transformed to P.pastoris X-33,and positive clones were screened with zeocin resistance for expression induced by 0.5% methanol.The expressed product was identified by SDS-PAGE,and antibacterial activity was determined by agarose gel thin plates seeded with Escherichia coli DH5α.Results pPICZα-A-CecP1 was successfully transfected into P.pastoris X-33,and cecropin P1 with a relative molecular mass of about 6.3 was expressed.It showed antibacterial activity to Gram-negative E.coli DH5α.Conclusion Cecropin P1 was successfully expressed in P.pastoris,and the cecropin P1 protein exhibited antibacterial activity.