Yue Lixi, Chi Zhenming, Wang Lin, Liu Jia, Madzak Catherine, Li Jing, Wang Xianghong
Unesco Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road No. 5, Qingdao, China.
J Microbiol Methods. 2008 Feb;72(2):116-23. doi: 10.1016/j.mimet.2007.11.011. Epub 2007 Nov 22.
In this study, a new surface display plasmid (pINA1317-YlCWP110) was constructed in Yarrowia lipolytica using C-terminal anchor domain of YlCWP1 from Y. lipolytica based on plasmid pINA1317, a pre-existing auto-cloning system for heterologous protein production in Y. lipolytica. When the genes encoding enhanced green fluorescent protein (EGFP) and haemolysin derived from the bacterium Vibrio harveyi were cloned into the newly constructed surface display plasmid, respectively, and expressed in cells of Y. lipolytica, we found that the target proteins were successfully displayed on the yeast cells and 100% of the yeast cell had anchoring target proteins. It was also shown that the yeast cells displaying haemolysin had haemolytic activity towards erythrocytes from flounder, indicating that the fusion protein remained functional. Therefore, the newly constructed surface display plasmid will have many applications in different fields such as in immobilized biocatalyst, bioconversion, bioremediation, live vaccine development and ultra-high-throughput screening for the identification of novel biocatalysts because it has many unique characteristics. To our knowledge, this work constitutes the first report of a surface display expression system in Y. lipolytica.
在本研究中，基于解脂耶氏酵母中预先存在的用于异源蛋白生产的自克隆系统质粒pINA1317，利用解脂耶氏酵母YlCWP1的C端锚定结构域在解脂耶氏酵母中构建了一种新的表面展示质粒（pINA1317-YlCWP110）。当将编码增强型绿色荧光蛋白（EGFP）和源自哈维氏弧菌的溶血素的基因分别克隆到新构建的表面展示质粒中，并在解脂耶氏酵母细胞中表达时，我们发现目标蛋白成功展示在酵母细胞上，且100%的酵母细胞都锚定有目标蛋白。还表明展示溶血素的酵母细胞对牙鲆红细胞具有溶血活性，这表明融合蛋白仍具有功能。因此，新构建的表面展示质粒将在固定化生物催化剂、生物转化、生物修复、活疫苗开发以及用于新型生物催化剂鉴定的超高通量筛选等不同领域有许多应用，因为它具有许多独特特性。据我们所知，这项工作构成了解脂耶氏酵母中表面展示表达系统的首次报道。
