Protein Expression and Mass Spectrometry

蛋白质表达和质谱分析

基本信息

批准号：
7480032
负责人：
Charles Scott Craik
金额：
$ 41.49万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-01 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7480032
关键词：
Address Affinity Affinity Chromatography Automation Baculoviruses Binding Biochemical Bioinformatics Cell physiology Cell-Free System Cells Cloning Codon Nucleotides Complex Coupled Crystallization Cultured Cells Cytolysis DNA repair protein Data Development Differential Scanning Calorimetry Dissociation Ensure Escherichia coli Foundations Gel Chromatography Genes HIV Human Insecta Kinetochores Length Ligase Mammalian Cell Mass Spectrum Analysis Measures Membrane Proteins Messenger RNA Methods N-terminal Nucleic Acids Numbers Organism Physiological Post-Translational Protein Processing Procedures Process Production Proteins Proteolysis Proteome Proteomics Protocols documentation Publishing RNA Splicing Rate Recombinant Proteins Recombinants Refractory Reporting Resources Ribosomal RNA Robotics Screening procedure Solubility Spectrometry, Mass, Electrospray Ionization Surface Surface Plasmon Resonance System Technology Tertiary Protein Structure Testing Transcriptional Regulation Two-Hybrid System Techniques Yeasts design desire expression cloning genetic regulatory protein intracellular protein transport light scattering monomethoxypolyethylene glycol mutant novel protein degradation protein expression protein protein interaction protein transport scale up stoichiometry success

项目摘要

Development of a high-throughput recombinant protein expression pipeline and methods to analyze and validate physiologically assembled complexes are essential for the success of the HARC Center structural efforts. As a starting point, it is important to carefully re-examine the physiological relevance of a reported interaction, as many have been initially identified using over-expression systems such as yeast two-hybrid assays, that can result in unnatural, 'nonspecific' binding at high concentration. The 'completeness1 of any given complex also is an issue, particularly for structural studies where each macromolecular interface may contribute to complex stability and surfaces may be remodeled by the interactions. Tandem affinity purification strategies coupled with mass spectrometry have been used successfully on many proteins in multiple organisms. Much of these data have been generated using S. cerevisiae where mid-scale proteomics studies have defined machinery involved in various cellular processes, including kinetochore function (353, 354), mRNA splicing (355, 356), and transcriptional regulation (357). Krogan has used this approach extensively to identify novel complexes and new components of previously characterized complexes involved in transcriptional regulation, rRNA processing, mRNA splicing, protein degradation, DNA repair, and protein trafficking (358-378) and has developed cutting-edge experimental and computational technology.

高通量重组蛋白表达管道和方法的开发分析和验证生理组装复合物对于 HARC 的成功至关重要中心结构努力。作为起点，重要的是仔细重新检查生理学所报告的相互作用的相关性，因为许多相互作用最初是使用过度表达系统识别出来的例如酵母双杂交检测，可能会导致高浓度下不自然的“非特异性”结合。任何给定复合体的“完整性”也是一个问题，特别是对于结构研究，其中每个大分子界面可能有助于复杂的稳定性，并且表面可以通过互动。串联亲和纯化策略与质谱联用已被使用成功地作用于多种生物体中的许多蛋白质。这些数据大部分是使用 S 生成的。酿酒酵母，其中中等规模的蛋白质组学研究已经定义了参与各种细胞的机制过程，包括动粒功能 (353, 354)、mRNA 剪接 (355, 356) 和转录监管（357）。 Krogan 广泛使用这种方法来识别新的复合物和新的先前表征的参与转录调控的复合物的成分，rRNA 加工、mRNA 剪接、蛋白质降解、DNA 修复和蛋白质运输 (358-378) 开发了尖端的实验和计算技术。