HIGH-THROUGHPUT CRYSTALLIZATION OF PROTEINS

Emerald BioStructures，Inc.正在开发一套完整的结晶 专为高通量结晶设计的研究工具和软件 由机器人以及个人研究人员使用的标准实验室使用 很少或没有以前的结晶研究经验。这些工具提供了 确定结晶条件的简化和系统化方法 产生X射线衍射级质量的蛋白质晶体。在此应用程序中， 调查人员建议开发一种系统的和普遍适用的方法 用于制备10s毫克的纯化蛋白 直接放入他们的集成结晶包中，从而特别 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)， Invitgen Corporation(表达就绪基因的构建和验证 风暴载体)和Emerald BioStructure，Inc.(结晶 试剂和软件，以及X射线结构测定)。我们的目标是 自动化全长靶蛋白的生产和结晶 未知的结构或功能未知的 这是因为他们的新型晶体监视器的可用性 能够高速收集结晶试验数据的软件 使用语音识别，这将被编程为驱动定制 结晶机器人。我们将利用Invitgen雄心勃勃的努力 生成表达就绪的GeneStorm载体，编码所有人类开放 读框(ORF)。分子模拟公司将使用他们的基因图谱 识别目标人类ORF的软件，其结构确定为 预计将产生全新的结构信息。这些公司联合在一起 将探索系统化程序的效用，以生产、净化和 结晶在E.Coli或昆虫细胞中表达的可溶性天然或复性蛋白质。 此外，他们还提出了一种新的复性和结晶过程。 蛋白质在相同的蒸气扩散腔中。一种或多种新奇的结晶 全长蛋白质将确定他们方法的可行性，从而 在后续阶段实现更完全自动化的结构基因组学工作 研究的成果。 建议的商业应用： 如果成功，这个第一阶段项目将形成一个自动化系统的基础 用于蛋白质结晶。因此，拟议的研究将有助于- 通过X射线结构测定蛋白质，并有助于孕育出新的 大分子结晶学的服务业。

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.