不同信号肽对胞外β-(1,3)-葡聚糖酶在巴斯德毕赤酵母中表达的影响  

Influence of Different Signal Peptides on Exo -β -(1,3) -Glucanase Expressed in Pichia pastoris

作　　者：杜济良[1] 赵洪亮[1] 薛冲[1] 任敏[1] 刘志敏[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2010年第6期798-802,837,共6页Letters in Biotechnology

摘　　要：目的:在胞外β-(1,3)-葡聚糖酶成熟肽的N端添加不同类型的信号肽,研究不同信号肽对其在巴斯德毕赤酵母中表达水平的影响。方法:通过融合PCR方法将α成熟交配因子(MFα)、成熟交配因子Pre肽(αPre)和共翻译转运信号肽(Bip信号肽)等3种信号肽的DNA片段连接至胞外β-(1,3)-葡聚糖酶成熟肽基因的5′端,利用PCR扩增包含其自身信号肽的胞外β-(1,3)-葡聚糖酶基因,将上述4种基因片段分别插入pPIC9质粒,再转化巴斯德毕赤酵母GS115宿主菌并诱导表达;测定胞外β-(1,3)-葡聚糖酶的活性,检测其表达水平。结果:目前在毕赤酵母中已广泛使用的MFα信号肽介导的胞外β-(1,3)-葡聚糖酶表达水平最高,其次是αPre,Bip信号肽介导的该酶表达水平是酶自身信号肽介导的表达水平的2倍。结论:共转运信号肽能够提高胞外β-(1,3)-葡聚糖酶的表达水平。Objective： To explore the influence of different signal peptides on the expression level of exo-β-（1,3）-glucanase in Pichia pastoris.Methods： The DNA fragments of three different signal peptides,α mature factor（MFα）,αPre and Bip,were linked to the gene of the mature peptide of exo-β-（1,3）-glucanase at its 5′ end by fusion PCR,then those gene fragments and the full length exo-β-（1,3）-glucanase gene were inserted into plasmid pPIC9 respectively to construct four different expression plasmids.Following the plasmids were transformed into P.pastoris GS115 respectively,the exo-β-（1,3）-glucanase were expressed by methanol induction.The exoglucanase activity was determined to evaluate the exoglucanase＇s expression level.Results： The recombinant exo-β-（1,3）-glucanase with the widely used signal leading peptide MFα was expressed at the highest level,and that with αPre signal peptide was the next.Meanwhile,compared with the glucanase led by itself signal peptide,the glucanase led by the cotranslation translocation signal peptide Bip reached the double expression level.Conclusion： The cotranslation translocation can increase the expression level of the exo-β-（1,3）-glucanase.

关 键 词：β-(1 3)-葡聚糖酶 信号肽 共翻译转运 巴斯德毕赤酵母 

分 类 号：Q75[生物学—分子生物学] Q78