Tanino Takanori, Fukuda Hideki, Kondo Akihiko
Division of Molecular Science and Material Engineering, Graduate School of Science and Technology, and Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, Nada-ku, Japan.
Biotechnol Prog. 2006 Jul-Aug;22(4):989-93. doi: 10.1021/bp060133+.
A Pichia pastoris cell-surface display system was constructed using a Flo1p anchor system, which was developed in Saccharomyces cerevisiae. The lipase from Rhizopus 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 degrees C was not only stable but also increased to about 6.5 times the initial value after 4 h incubation.
利用在酿酒酵母中开发的Flo1p锚定系统构建了毕赤酵母细胞表面展示系统。将带有前导序列的米根霉脂肪酶（ProROL）用作模型蛋白，并将其与由Flo1p的1-1099位氨基酸组成的锚定蛋白（FS锚定蛋白）进行基因融合。所得融合蛋白FSProROL在醇氧化酶1启动子（pAOX1）的控制下表达。对免疫标记的毕赤酵母细胞进行荧光显微镜观察发现ProROL展示在细胞表面，蛋白质印迹分析表明融合蛋白FSProROL非共价连接到细胞壁且高度糖基化。毕赤酵母细胞的脂肪酶活性受诱导阶段甲醇浓度的影响。令人惊讶的是，在60℃孵育的细胞上展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。
