Secretion expression of human alpha-defensin 5 in Pichia pastoris

Objective To explore the feasibility of high-level secretion expression of bioactive mature human alpha-defensin 5 (mHD5) in Pichia pastoris Methods DNA fragment containing mHD5 coding sequence with biased codons of Pichia pastoris was amplified by PCR,then inserted into the Pichia pastoris expression vector pPIC9K After the recombinant plasmid was transformed into the yeast host strain GS115 by electroporation,the transformants with multi-copy inserts were selected by PCR identification and G418 phenotype screen After optimization of the flask-shaking culture fermentation,the expression of recombinant mHD5 (rmHD5) induced by methanol was analyzed by Tricine-SDS-PAGE and Western blot The bioactivity of rmHD5 was examined by antibacterial assay Results The plasmid pPIC9K-mHD5 was correctly constructed Three transferred GS115 strains containing multi-copy mHD5 gene were singled out,which could grow in the media with 8 mg/ml G418 and showed His+/Mut+ phenotype The yield of rmHD5 was about 120 mg/L in the clarified broth of fermentation media The rmHD5 displayed obvious antibacterial activity against both EC25922 and SA25923 Conclusion The strategy of secretion expression of defensin gene in Pichia pastoris may be an effective way to produce bioactive antimicrobial peptide on a large scale