Mini-Chromosome and Fluorescent Proteins for Expression of Protein Complexes

用于蛋白质复合物表达的微型染色体和荧光蛋白

• 批准号: 8248457
• 负责人: Ronald Godiska
• 金额: $ 23.08万
• 依托单位: LUCIGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-21 至 2014-03-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8248457
• 关键词: 3-Dimensional; Binding; Biological Assay; Biology; C-terminal; Cell Culture Techniques; Cell membrane; Cells; Cellular Structures; Chromosomes; Cloning; Code; Codon Nucleotides; Complex; Cytoplasm; Detection; Development; Disease; DsRed; Escherichia coli; Evaluation; Fluorescence; Fluorescence Resonance Energy Transfer; Gel; Gene Proteins; Genes; Goals; Government; Green Fluorescent Proteins; Human Herpesvirus 4; Image; Image Analysis; Individual; Libraries; Maintenance; Messenger RNA; Monitor; N-terminal; Nuclear; Operon; Oranges; Organism; Oxygen; Peptide Signal Sequences; Peptides; Persons; Phase; Phytochrome; Plasmids; Post-Translational Protein Processing; Property; Protein Complex Subunit; Proteins; Recombinant Proteins; Recombinants; Replication Origin; Reporter; Research; Research Personnel; Sampling; Signal Transduction; Small Business Innovation Research Grant; Solubility; Spectrum Analysis; Structure; System; Testing; Time; Tissues; VP 16; Variant; Viral; Work; Yeasts; base; commercialization; cost; expression vector; fluorophore; improved; in vivo; light scattering; mutant; novel; promoter; protein complex; protein expression; protein protein interaction; tool; vector

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.

描述（由申请人提供）：多亚基蛋白复合物的稳健表达对于理解生物体的基本生物学至关重要。大肠杆菌或酵母中各个成分的表达可能快速且具有成本效益，但是在这些宿主中表达时，许多哺乳动物蛋白的表达或溶解度较差。此外，它们缺乏适当的翻译后修饰，这对于与其他蛋白质的适当活动或相互作用可能至关重要。当前的哺乳动物表达系统通常每质粒表达一种蛋白质，维持在细胞质中或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。监测和分析由重组蛋白形成的复合物的进一步并发症。因此，该阶段I建议的目的是开发一个系统，以在稳定的“迷你染色体”上有条件地表达多种蛋白质作为哺乳动物操纵子。表达矢量将基于线性“ Pjazz”载体，该载体消除了克隆的功能和结构障碍。此外，我们将以红色和近红外荧光团的形式开发新型细菌植物色素（物理），以监测和量化蛋白质之间的短暂相互作用。这些物理可用于体内和变性凝胶的荧光成像。此外，它们将具有FRET功能。该系统将对多亚基络合物中蛋白质的结构，功能，组装和相互作用进行广泛的研究。它还将促进结合伴侣的鉴定和改进的细胞结构成像。 公共卫生相关性：细胞中的大量蛋白质形成独特的多生成络合物，通常很难研究。我们建议开发一个新型系统，以便于细胞培养物中的蛋白质复合物产生和监测。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。