通过生长素诱导降解结构域（AID）融合快速消耗出芽酵母蛋白质

Rapid Depletion of Budding Yeast Proteins via the Fusion of an Auxin-Inducible Degron (AID).

作者信息

Nishimura Kohei, Kanemaki Masato T

机构信息

Center of Frontier Research, National Institute of Genetics, Research Organization of Information and Systems, Shizuoka, Japan.

出版信息

Curr Protoc Cell Biol. 2014 Sep 2;64:20.9.1-16. doi: 10.1002/0471143030.cb2009s64.

DOI:10.1002/0471143030.cb2009s64

PMID:25181302

Abstract

The auxin-inducible degron (AID) system allows the rapid and reversible proteolysis of proteins of interest, and enables the generation of conditional mutants of budding yeast. The construction of budding yeast AID mutants is simple, and the effect of depletion of essential proteins on proliferation can be confirmed by analyzing their phenotype. In this protocol, we describe a procedure to generate AID mutants of budding yeast via a simple transformation using PCR-amplified DNA. We also describe methods to confirm the depletion of proteins of interest that are required for proliferation by serial-dilution and liquid-culture assays.

摘要

生长素诱导降解子（AID）系统能够实现对目标蛋白质的快速且可逆的蛋白水解，并能够构建出芽酵母的条件性突变体。构建出芽酵母AID突变体的方法很简单，通过分析其表型可以确认必需蛋白质缺失对增殖的影响。在本实验方案中，我们描述了一种通过使用PCR扩增的DNA进行简单转化来生成出芽酵母AID突变体的程序。我们还描述了通过系列稀释和液体培养试验来确认增殖所需的目标蛋白质缺失的方法。