King Chih-Yen, Wang Hui-Ling, Chang Hsiang-Yu
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.
Methods. 2006 May;39(1):68-71. doi: 10.1016/j.ymeth.2006.04.003.
We present methods to prepare infectious Sup35 protein aggregates and use them for genetic transformation of yeast. The protein aggregates are prepared from bacterially expressed recombinant protein, which is converted to amyloid fibers by extended incubation or by nucleated growth using yeast prion particles as seeds. The aggregates are introduced into yeast by a modified spheroplast transformation protocol. The phenotype of the yeast transformants is further characterized by robust prion strain typing methods. The methodology can be used to introduce different [PSI(+)] particles to many laboratory yeast genetic backgrounds. It can be adapted for applications in other yeast prion systems as well.
我们展示了制备传染性Sup35蛋白聚集体并将其用于酵母基因转化的方法。蛋白聚集体由细菌表达的重组蛋白制备而成,通过长时间孵育或使用酵母朊病毒颗粒作为种子进行成核生长,将其转化为淀粉样纤维。通过改良的原生质球转化方案将聚集体导入酵母。通过强大的朊病毒菌株分型方法进一步表征酵母转化体的表型。该方法可用于将不同的[PSI(+)]颗粒引入许多实验室酵母遗传背景。它也可适用于其他酵母朊病毒系统的应用。