Construction of a Pichia pastoris Cell‐Surface Display System Using Flo1p Anchor System
Construction of a Pichia pastoris Cell‐Surface Display System Using Flo1p Anchor System
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DOI：
10.1021/bp060133
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发表时间：
2006
影响因子：
2.9
通讯作者：
Takanori Tanino;H. Fukuda;A. Kondo
中科院分区：
文献类型：
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作者：
Takanori Tanino;H. Fukuda;A. Kondo
使用在酿酒酵母中开发的Flo 1 p锚系统构建了巴斯德毕赤酵母细胞表面展示系统。以具有前体序列的根霉脂肪酶（ProROL）为模型蛋白，将其与Flo 1 p的1-1099位氨基酸组成的锚（FS锚）基因融合。所得融合蛋白FSProROL在醇氧化酶1启动子（pAOX 1）的控制下表达。免疫标记的巴斯德毕赤酵母细胞的荧光显微镜显示ProROL展示在细胞表面，Western blot分析显示融合蛋白FSProROL非共价连接于细胞壁并高度糖基化。甲醇浓度对毕赤酵母细胞的脂肪酶活性有影响。令人惊讶的是，在60 °C孵育的细胞上显示的脂肪酶活性不仅稳定，而且在孵育4小时后增加到初始值的约6.5倍。
A Pichia pastoris cell‐surface display system was constructed using a Flo1p anchor system, which was developed in Saccharomyces cerevisiae. The lipase fromRhizopus oryzae with a pro sequence (ProROL) was used as the model protein and was genetically fused to the anchor consisting of amino acids 1–1099 of Flo1p (FS anchor). The resulting fusion protein FSProROL was expressed under the control of the alcohol oxidase 1 promoter (pAOX1). The fluorescence microscopy of immunolabeled P. pastoris cells revealed that ProROL was displayed on the cell surface, and Western blot analysis revealed that the fusion protein FSProROL was noncovalently attached to the cell wall and highly glycosylated. The lipase activity of P. pastoris cells was affected by the methanol concentration for the induction phase. Surprisingly, the activity of lipase displayed on the cells incubated at 60 °C was not only stable but also increased to about 6.5 times the initial value after 4 h incubation.