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具有关键重要性。然而，需要开发更强大的方法来（1）验证潜在的相互作用伴侣，（2）有效筛选复合物的形成，以及（3）产生足够量的功能蛋白质复合物用于深入的功能和结构研究。为了满足这些需求，我们将（1）开发新的基于荧光的高通量筛选来评估蛋白质-蛋白质相互作用;（2）开发快速筛选方法来区分瞬时和稳定相互作用的伴侣。(3)最后，由于蛋白质复合物的功能表征和结构测定取决于以高产量重组产生它们的能力，我们将开发一种多蛋白质表达系统，该系统基于其表达水平与每个蛋白质组分相关的不同荧光标记。总之，这些方法提供了用于验证、表征和产生蛋白质复合物以用于结构和功能研究的高通量管道。这个平台，以及在这个提议下开发的相关试剂，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质复合物的科学家。

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.