HIGH-THROUGHPUT CRYSTALLIZATION OF PROTEINS

蛋白质的高通量结晶

• 批准号: 6143971
• 负责人: LANCE J STEWART
• 金额: $ 23.06万
• 依托单位: EMERALD BIOSTRUCTURES
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6143971
• 关键词: computer program /software; computer simulation; crystallization; functional /structural genomics; high throughput technology; molecular cloning; nucleic acid sequence; open reading frames; protein purification; protein structure; robotics; technology /technique development; transfection /expression vector

Emerald BioStructures, Inc. Is developing an integrated set of crystallization research tools and software that are designed for high-throughput crystallization by robots as well as the standard laboratory use by individual researchers with little or no prior crystallization research experience. These tools provide a streamlined and systematic approach for identifying crystallization conditions that yield X-ray diffraction quality protein crystals. In this application, the investigators propose to develop a systematic and generally applicable method for the preparation of 10s of milligrams of purified proteins that will fed directly into their integrated crystallization package, thereby specifically addressing the rate-limiting aspects of the Structural Genomics Initiative. We propose to fuse the complementary expertise of Molecular Simulations, Inc. (Protein structure analysis and target selection using GeneAtlas software), Invitrogen Corporation (construction and validation of expression-ready Gene Storm vectors) and, with those of Emerald BioStructure, Inc. (Crystallization reagents and software, and X-ray structure determination). Our goal is to automate the production and crystallization of full-length target proteins of unknown structure or function. This is made possible by the availability of their novel Crystal Monitor software which enables high speed collection of crystallization trial data using speech recognition, which will be programmed to drive custom built crystallization robots.We will take advantage of Invitrogen's ambitious efforts to generate expression ready GeneStorm vectors encoding all human open reading frames (ORF's). Molecular Simulations, Inc., will employ their GeneAtlas software to identify target human ORFs whose structure determinations are expected to yield completely novel structural information. Together the companies will explore the utility of systematic procedures for producing, purifying, and crystallizing soluble native or refolded proteins expressed in E. Coli or insect cells. In addition, they propose an innovative procedure for renaturing and crystallizing proteins in the same vapor diffusion chamber. Crystallization of one or more novel full-length proteins will establish the feasibility of their approach, thereby enabling a more fully automated Structural Genomics effort in subsequent phases of research. PROPOSED COMMERCIAL APPLICATION: If successful, this Phase I project would form the basis for an automated system for protein crystallization. Hence, the proposed research will facilitate the high- throughput X-ray structure determination of proteins, and help to spawn a new service industry in macromolecular crystallography.

Emerald BioStructures，Inc.正在开发一套综合的结晶 专为高通量结晶而设计的研究工具和软件 机器人以及个人研究人员的标准实验室使用， 很少或没有结晶研究经验。这些工具提供了一个 用于确定结晶条件的简化和系统的方法 产生X射线衍射质量的蛋白质晶体。在本申请中， 研究人员建议开发一种系统的、普遍适用的方法， 用于制备10毫克的纯化蛋白质， 直接集成到其集成结晶包装中， 解决结构基因组学倡议的限速问题。 我们建议融合分子模拟公司的互补专业知识。 （使用GeneAtlas软件进行蛋白质结构分析和靶标选择）， Invitrogen Corporation（表达就绪基因的构建和验证 风暴矢量），并与那些翡翠生物结构公司。（结晶） 试剂和软件，以及X射线结构测定）。我们的目标是 使全长靶蛋白的生产和结晶自动化， 未知的结构或功能。 这是可能的，他们的新颖的水晶显示器的可用性 能够高速收集结晶试验数据的软件 使用语音识别，这将被编程，以驱动定制的 结晶机器人。我们将利用英杰公司雄心勃勃的努力， 以产生表达准备GeneStorm载体编码所有人类开放 阅读框（ORF）。分子模拟公司，将使用他们的基因图谱 软件来鉴定靶人类ORF，其结构测定被 有望产生全新的结构信息。公司一起 将探索系统程序的效用，用于生产，纯化， 结晶大肠杆菌中表达的可溶性天然或重折叠蛋白质。或者昆虫细胞。 此外，他们还提出了一种创新的复性和结晶方法， 蛋白质在同一个蒸汽扩散室。结晶一种或多种新颖的 全长蛋白质将建立他们的方法的可行性，从而 使结构基因组学工作在后续阶段更加自动化 的研究。 拟定商业应用： 如果成功，第一阶段项目将成为自动化系统的基础 用于蛋白质结晶。因此，拟议的研究将促进高- 通过X射线测定蛋白质的结构，并帮助产生一个新的 高分子晶体学服务行业。