Sato N, Matsumoto T, Ueda M, Tanaka A, Fukuda H, Kondo A
Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Japan.
Appl Microbiol Biotechnol. 2002 Dec;60(4):469-74. doi: 10.1007/s00253-002-1121-6. Epub 2002 Oct 24.
We investigated the influence of anchor length on the reactivity to polymer substrate of enzyme displayed on yeast cell surfaces. Using various lengths [42, 102, 146, 318, 428, and 1,326 amino acids (aa)] of the C-terminal region of the Saccharomyces cerevisiae Flo1 protein (Flo1p), which plays a major role in yeast flocculation, six display systems with various anchor lengths were constructed. In these systems, the target protein was displayed on the yeast cell surface under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis ( UPR-ICL). Cell-surface display of Rhizopus oryzae glucoamylase by these systems was induced and confirmed in all systems by immunofluorescence microscopy and immunoblotting. Flow-cytometer measurement of the fluorescence intensity of immunofluorescence-labeled yeast cells displaying glucoamylase indicated that glucoamylase displayed with longer anchors, especially those of 428 and 1,326 aa in length, had higher reactivity to antibodies. The reactivity of starch to displayed glucoamylase, which was evaluated by plate assay, increased with anchor length, as did the cell growth-rate in starch-containing medium. These results indicate that cell-surface display systems using 428- and 1,326-aa length anchors of Flo1p are effective for the display of enzymes on the outer surface of yeast cells.
我们研究了锚定长度对展示在酵母细胞表面的酶与聚合物底物反应性的影响。利用在酵母絮凝中起主要作用的酿酒酵母Flo1蛋白（Flo1p）C端区域的不同长度[42、102、146、318、428和1326个氨基酸（aa）]，构建了六种具有不同锚定长度的展示系统。在这些系统中，目标蛋白在热带假丝酵母异柠檬酸裂合酶基因5'上游区域（UPR-ICL）的控制下展示在酵母细胞表面。通过免疫荧光显微镜和免疫印迹在所有系统中诱导并确认了这些系统对米根霉葡糖淀粉酶的细胞表面展示。对展示葡糖淀粉酶的免疫荧光标记酵母细胞的荧光强度进行流式细胞仪测量表明，展示有较长锚定物（尤其是长度为428和1326 aa的锚定物）的葡糖淀粉酶对抗体具有更高的反应性。通过平板试验评估的淀粉对展示的葡糖淀粉酶的反应性随锚定长度增加，在含淀粉培养基中的细胞生长速率也如此。这些结果表明，使用长度为428和1326 aa的Flo1p锚定物的细胞表面展示系统对于在酵母细胞外表面展示酶是有效的。
