人α-防御素5前体蛋白在毕赤酵母中分泌表达的研究

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

为了探索人α-防御素5(HD5)前体蛋白基因在酵母中高效分泌表达的可行性,将其对应基因克隆到载体pPIC9K,得到重组载体pPIC9K-preproHD5,经SacI线性化后,转入Pichiapastoris GS115,得到重组毕赤酵母菌GS115/pPIC9K-preproHD5。应用PCR技术筛选高拷贝转化株,用BMGY培养GS115/pPIC9K-HD5至OD600为2～6时,用BMMY诱导培养120h,离心取上清液,用Tricien-SDS-PAGE和蛋白定量试剂盒分析人α-防御素5前体蛋白的表达量。琼脂扩散法检测人α-防御素5前体蛋白的抑菌活性,发现其对金黄色葡萄球菌(ATCC6538)和大肠杆菌(ATCC10231)2种标准菌株具明显的抑菌活性。人α-防御素5前体基因可在毕赤酵母实现分泌型表达,该策略可能成为防御素工业化开发的有效途径。

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.