Fluorescence methods for HT validation and production of protein complexes

描述（由申请人提供）：大分子复合物在几乎所有生命过程中都是至关重要的。然而，需要开发更强大的方法来(1)验证潜在的相互作用伙伴，(2)有效筛选复合物形成，以及(3)产生足够数量的功能性蛋白质复合物以进行深入的功能和结构研究。为了满足这些需求，我们将(1)开发新的高通量荧光筛选来评估蛋白质-蛋白质相互作用；(2)建立快速筛选方法，区分瞬态和稳定的相互作用伙伴。(3)最后，由于蛋白质复合物的功能表征和结构确定取决于其高产重组生产的能力，我们将开发一种基于不同荧光标记的多蛋白表达系统，其表达水平与每种蛋白质成分相关。总之，这些方法为验证、表征和生产用于结构和功能研究的蛋白质复合物提供了高通量管道。该平台以及根据该提案开发的相关试剂将提供一个急需的工具箱，这将极大地帮助任何研究真核蛋白复合物的科学家。

DESCRIPTION (provided by applicant): Macromolecular complexes are of key importance in virtually all life processes. However, more powerful methods need to be developed to (1) validate potential interacting partners, (2) efficiently screen for complex formation, and (3) produce sufficient amounts of functional protein complexes for in depth functional and structural studies. To address these needs we will (1) develop new high-throughput fluorescence-based screens to assess protein-protein interactions; (2) develop rapid screening method to distinguish between transient and stably interacting partners. (3) Finally, since functional characterization and structure determination of protein complexes depends on the capability to produce them recombinantly in high yield, we will develop a multi-protein expression system, based on distinct fluorescent markers whose expression levels are correlated with each protein component. Together, these methods provide a high- throughput pipeline for validating, characterizing, and producing protein complexes for structural and functional studies. This platform, and the associated reagents developed under this proposal, will provide a much needed toolbox that will greatly aid any scientist studying eukaryotic protein complexes.