Secreted Expression of Human alpha-defensin-5 Preproprotein in P ichia pastoris

To explore the feasibility of high-level secretion expression of bioactive human alpha-defensin-5 preproprotein(preproHD5) in Pichia pastoris,DNA fragment containing preproHD5 was inserted into the yeast expression vector pPIC9K successfully.The recombinant vector pPIC9K-preproHD5 was linearized with enzyme SacI and was transformated into Pichia pastoris GS115.The Transformants were cultured with BMGY medium and induced with BMMY medium since the cell density reached an OD600=2～6.The recombinant protein could be detected in the cultured supernatant by Tricien-SDS-PAGE and protein assay reagents shows that the yield of preproHD5 was about 200mg/L in the clarified broth of fermentation media.Plate test indicated that the supematant could inhibite the growth of Escherichia coli ATCC6538 and Staphylococcus aureus ATCC10231.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.