Mini-Chromosome and Fluorescent Proteins for Expression of Protein Complexes

描述(由申请人提供)：多亚基蛋白质复合体的强健表达对于理解生物体的基本生物学至关重要。单个组分在大肠杆菌或酵母中的表达可能是快速和经济的，但许多哺乳动物蛋白在这些宿主中表达时表现出较差的表达或溶解性。此外，它们缺乏适当的翻译后修饰，这可能是正常活性或与其他蛋白质相互作用所必需的。目前的哺乳动物表达系统通常在每个质粒上表达一种蛋白质，要么保持在细胞质中，要么随机整合到染色体上。这些蛋白质的稳定和协调表达是不可能的。在监测和分析重组蛋白形成的复合体时，会出现更多的复杂情况。因此，这一第一阶段提案的目标是开发一种系统，将多种蛋白质作为哺乳动物操纵子有条件地表达在稳定的“小染色体”上。表达载体将基于线性“pJAZZ”载体，这消除了克隆的功能和结构障碍。此外，我们将开发新型细菌光敏色素(Phys)作为红色和近红外荧光团，以监测和量化蛋白质之间的瞬时相互作用。这些PHY将在体内和变性凝胶中用于荧光成像。此外，他们将拥有FRET能力。这一系统将使对多亚基复合体中蛋白质的结构、功能、组装和相互作用的广泛研究成为可能。它还将有助于识别结合伙伴和改进细胞结构的成像。 与公共卫生相关：细胞内的大量蛋白质形成独特的多亚单位复合体，这通常很难研究。我们建议开发一种新的系统来容易地在细胞培养中生产和监测蛋白质复合体。这项工作将使研究人员能够更好地了解正常和疾病情况下的蛋白质相互作用。

DESCRIPTION (provided by applicant): Robust expression of multi-subunit protein complexes is crucial for understanding the basic biology of organisms. Expression of the individual components in E. coli or yeast may be quick and cost-efficient, but many mammalian proteins show poor expression or solubility when expressed in these hosts. Further, they lack appropriate post-translational modifications, which may be essential for proper activity or interaction with other proteins. Current mammalian expression systems typically express one protein per plasmid, maintained either in the cytoplasm or randomly integrated onto the chromosome. Stable and coordinated expression of these proteins is not possible. Further complications arise in monitoring and analyzing complexes formed from the recombinant proteins. The goal of this Phase I proposal is therefore to develop a system to conditionally express multiple proteins as a mammalian operon on a stable "mini chromosome". The expression vector will be based on the linear "pJAZZ" vector, which removes functional and structural barriers to cloning. In addition, we will develop novel bacterial phytochromes (PHYs) as red and near-infrared fluorophores to monitor and quantify transient interactions among proteins. These PHYs will be useful for fluorescent imaging in vivo and in denaturing gels. Moreover, they will have FRET capability. This system will enable a wide range of studies on structure, function, assembly, and interaction of proteins in multi-subunit complexes. It will also facilitate identification of binding partners and improved imaging of cellular structures. PUBLIC HEALTH RELEVANCE: A large number of proteins within cells form unique multi-subunit complexes, which are typically very difficult to study. We propose to develop a novel system to readily produce and monitor protein complexes in cell cultures. This work will enable researchers to better understand protein interactions in normal and disease situations.