Multiplexed screening protein expression libraries

多重筛选蛋白表达文库

• 批准号: 6735392
• 负责人: WILLIAM D HARRIMAN
• 金额: $ 16.22万
• 依托单位: TRELLIS BIOSCIENCE, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6735392
• 关键词: biotechnology; gel electrophoresis; high throughput technology; immunoglobulin G; peptide library; protein protein interaction; technology /technique development

DESCRIPTION (provided by applicant): Developing maps of the protein-protein interaction network in human cells is crucial to understanding cancers and other human diseases that alter the regulatory state of normal cells. These maps seek to represent the individual protein-protein interactions that in combination lead to enzymatic regulation, cellular localization, or pathway facilitation. Global analysis of protein-protein interaction requires techniques, which query the interaction of a single protein with all of the proteins in the cell. Whether based on in vitro or in vivo assays, analysis is currently limited by the monoplex nature of most available technology. In this application, Trellis proposes to develop a highly multiplexed pangenomic library screen for performing library x library interaction assays. The screening technology adapts Trellis' microscopic multihued beads into multiplexed detectors of protein-protein interaction. Applied to filter lifts from high-density phage plaque replicas, the beads can provide a 100 x 10,000 interaction environment for quantitative analysis by a specialized reader, with potential to grow to 1,000 x 1,000,000. In preliminary studies, the company has demonstrated efficient and specific fluorescent bead binding to protein spotted on PVDF membrane filters. The size of the beads is selected to flow through the membrane without trapping and to bind with rapid kinetics. In addition, the company has demonstrated its ability to produce multiple levels of fluor staining for these nanobeads and to distinguish between bead populations by microscopic spectral analysis with an instrument suitable for conversion to an affordable plate reader. In Phase I, Trellis will (1) optimize reagents and protocols for fluorescent bead binding to membrane filters, (2) adapt an affordable commercial microscope platform to support spectral analysis of beads binding to phage plaque replicas, and (3) develop the first screening reagents for multiplexed detection of expressed proteins and use them to define protocols that can be applied on a large scale in Phase II. In addition to development of protein-protein network maps, the multiplex library screening technology will have application in development of recombinant antibodies, investigations of autoimmune disease and cancers, and analysis of peptide recognition motifs.

描述（由申请人提供）：开发人类细胞中蛋白质-蛋白质相互作用网络的图谱对于理解改变正常细胞调节状态的癌症和其他人类疾病至关重要。这些图谱试图代表单独的蛋白质-蛋白质相互作用，这些相互作用结合在一起导致酶调节、细胞定位或通路促进。蛋白质相互作用的全局分析需要查询细胞中单个蛋白质与所有蛋白质的相互作用的技术。无论是基于体外还是体内测定，分析目前都受到大多数可用技术的单一性质的限制。 在本申请中，Trellis提出开发高度多重泛基因组文库筛选，用于进行文库X文库相互作用测定。该筛选技术将Trellis的显微多色珠应用于蛋白质-蛋白质相互作用的多重检测器。应用于高密度噬菌体噬菌斑复制品的过滤提升，珠粒可以提供100 x 10，000的相互作用环境，用于专业阅读器的定量分析，并有可能增长到1，000 x 1，000，000。 在初步研究中，该公司已经证明了高效和特异性的荧光珠与PVDF膜过滤器上的蛋白质结合。选择珠粒的尺寸以流过膜而不截留并以快速动力学结合。此外，该公司已证明其能够为这些纳米珠产生多个水平的荧光染色，并通过显微光谱分析区分珠群，该仪器适合转换为负担得起的酶标仪。 在第一阶段，Trellis将（1）优化荧光珠与膜过滤器结合的试剂和方案，（2）采用负担得起的商业显微镜平台，以支持珠与噬菌体噬菌斑复制品结合的光谱分析，以及（3）开发第一种用于表达蛋白质多重检测的筛选试剂，并使用它们来定义可以在第二阶段大规模应用的方案。 除了蛋白质-蛋白质网络图谱的开发之外，多重文库筛选技术将在重组抗体的开发、自身免疫性疾病和癌症的研究以及肽识别基序的分析中具有应用。