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.

翡翠生物结构，Inc。正在开发一组集成的结晶集 专为高通量结晶而设计的研究工具和软件 由机器人以及个别研究人员的标准实验室使用 几乎没有或没有以前的结晶研究经验。这些工具提供了 精简和系统的方法来识别结晶条件 产生X射线衍射质量蛋白晶体。在此应用程序中 调查人员建议开发一种系统的，通常适用的方法 为准备10毫克的纯化蛋白质，以喂养 直接进入其集成的结晶软件包，因此 解决结构基因组学计划的限速性方面。 我们建议融合Molecular Simulates，Inc。的互补专业知识。 （蛋白质结构分析和使用Geneatlas软件的目标选择）， Invitrogen Corporation（表达准备基因的构建和验证 风暴矢量）和与Emerald Biostructure Inc.（结晶化 试剂和软件以及X射线结构确定）。我们的目标是 自动化全长靶蛋白的生产和结晶 未知的结构或功能。 他们的新型水晶监视器的可用性使它成为可能 可以高速收集结晶试验数据的软件 使用语音识别，将编程以驱动自定义构建 结晶机器人。我们将利用Invitrogen的雄心勃勃的努力 生成表达式准备的基因斯托媒介，编码所有人类开放 读取帧（ORF）。分子模拟，Inc。将采用其基因 识别其结构确定的人类ORF的软件 预计将产生完全新颖的结构信息。共同的公司 将探索系统的程序生产，净化和 在大肠杆菌或昆虫细胞中表达的结晶可溶性天然或重折叠的蛋白质。 此外，他们提出了一种创新程序，以重新定性和结晶 同一蒸气扩散室中的蛋白质。一个或多个小说的结晶 全长蛋白将确定其方法的可行性，从而 在随后的阶段实现更全自动的结构基因组学工作 研究。 拟议的商业应用： 如果成功，此阶段I项目将构成自动化系统的基础 用于蛋白质结晶。因此，拟议的研究将促进高 吞吐量X射线结构的蛋白质测定，并有助于产生新的 大分子晶体学的服务业。