Rapid selection against truncation mutants in yeast reverse two-hybrid screens
Rapid selection against truncation mutants in yeast reverse two-hybrid screens
复制标题
DOI：
10.2144/01305st03
复制
发表时间：
2001-05-01
期刊：
影响因子：
2.7
通讯作者：
Huang, DCS
中科院分区：
文献类型：
--
作者：
Puthalakath, H;Strasser, A;Huang, DCS
酵母反向双杂交系统是一种强大的技术，用于分离蛋白质中的突变，消除其与已知伴侣的相互作用。选择是基于野生型相互作用赋予酵母细胞5-氟乳清酸（5-FOA）敏感性时施加的生长抑制的消除。这个系统的一个费力的组成部分是消除那些导致蛋白质截短的突变。通过将绿色荧光蛋白（GFP）融合到感兴趣的蛋白质动力蛋白轻链（LC 8）的C末端，我们能够快速分离不会导致蛋白质截短的突变。
The yeast reverse two-hybrid system is a powerful technique for isolating mutations in a protein that abolish its interaction with a known partner. Selection is based on abrogation of growth suppression imposed when wild-type interactions confer 5-fluoroorotic acid (5-FOA) sensitivity to yeast cells. A laborious component of this system is to eliminate those mutations that cause protein truncation. By fusing the green fluorescent protein (GFP) to the C-terminus of a protein of interest, dynein light chain (LC8), we were able to rapidly isolate mutations that did not result in protein truncation.