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强大表达对于理解生物体的基本生物学至关重要。在大肠杆菌或酵母中表达单个成分可能是快速和经济有效的，但许多哺乳动物蛋白质在这些宿主中表达时表现出较差的表达或溶解度。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每个质粒表达一种蛋白质，这些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物时出现了进一步的并发症。因此，本I期计划的目标是开发一种系统，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的操纵子。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，我们将开发新的细菌光敏色素（PHYs）作为红色和近红外荧光团来监测和量化蛋白质之间的瞬时相互作用。这些物理量将有助于荧光成像在体内和变性凝胶。此外，它们将具有FRET能力。该系统将能够广泛研究多亚基复合物中蛋白质的结构、功能、组装和相互作用。它还将促进结合伙伴的识别和改进细胞结构的成像。