Shibasaki Y, Kamasawa N, Shibasaki S, Zou W, Murai T, Ueda M, Tanaka A, Osumi M
Division of Material and Biological Sciences, Graduate School of Science, Japan Women's University, Bunkyo-ku, Tokyo, Japan.
FEMS Microbiol Lett. 2000 Nov 15;192(2):243-8. doi: 10.1111/j.1574-6968.2000.tb09389.x.
A starch-utilizing Saccharomyces cerevisiae strain was constructed by cell surface engineering. Distribution of the heterologous glucoamylase-alpha-agglutinin fusion protein on the yeast cell was analyzed by indirect fluorescence microscopy using an anti-glucoamylase antibody. Most of the intense fluorescence was first localized in the small bud, then observed on the entire cell wall of the daughter and mother cells. Fluorescence also accumulated at the neck region. These observations suggest that the display of the heterologous protein on the cell surface is carried with other cell wall components to the areas in which the cell wall is newly synthesized; the distribution is controlled by the cell cycle. Then, the heterologous protein-encoding gene was expressed in a sec1 mutant, in which secretory vesicles accumulate under restrictive temperature, and the produced protein was detected by immunoelectron microscopy. Most of the gold particles that reacted with the fusion protein were not localized in vesicles but in expanding endoplasmic reticulum. This phenomenon may be due to overproduction of the heterologous protein which was designed to be displayed on the cell wall. Artificial production of heterologous protein may have caused a relative shortage of glycosyl phosphatidylinositol anchors.
通过细胞表面工程构建了一株利用淀粉的酿酒酵母菌株。使用抗葡糖淀粉酶抗体，通过间接荧光显微镜分析了异源葡糖淀粉酶-α-凝集素融合蛋白在酵母细胞上的分布。大部分强烈荧光首先定位在小芽中，然后在子细胞和母细胞的整个细胞壁上观察到。荧光也在颈部区域积累。这些观察结果表明，异源蛋白在细胞表面的展示与其他细胞壁成分一起被携带到细胞壁新合成的区域；这种分布受细胞周期控制。然后，在sec1突变体中表达异源蛋白编码基因，在限制温度下分泌小泡会在其中积累，并通过免疫电子显微镜检测产生的蛋白质。与融合蛋白反应的大多数金颗粒不是定位在小泡中，而是定位在扩张的内质网中。这种现象可能是由于设计用于展示在细胞壁上的异源蛋白过量产生所致。异源蛋白的人工生产可能导致糖基磷脂酰肌醇锚相对短缺。
