The Cloning Protein Production, Crystallization, and X-ray Data Collection Core

克隆、蛋白质生产、结晶和X射线数据收集核心构成了一个分布式 X射线晶体结构测定流水线。该分布式结构利用了本地 专业知识，与德克萨斯农工大学的克隆设施，洛斯阿拉莫斯的蛋白质纯化设施 国家实验室，劳伦斯·利弗莫尔国家实验室的结晶设施，纳米结晶 位于加州大学伯克利分校的设施和位于高级光源的X射线数据收集和分析核心。 这个核心小组负责大部分的克隆、纯化、结晶和数据收集 与项目项目一起。这组核心实验室的总体目标是：1)提供快速访问 用于结构、生化和遗传学研究的可表达克隆文库，2)提供高度纯化的靶标 具有足够数量的蛋白质，通过标准化、自动化和 协议的并行化3)为晶体研究进行晶体筛选4)为以下项目提供筛选解决方案 抑制剂筛选，5)收集高分辨率MAD数据，每年求解30-40个结构。 管道设施能够使用改装的网关进行大规模克隆准备 克隆系统(Invitgen)，将可切割的融合标签整合到系统中，并允许 在大肠杆菌和耻垢分枝杆菌中均有表达。蛋白质生产设施具有生产6-8个蛋白质的能力 每周高纯度的目标蛋白，采用高产自动诱导发酵，平行细胞裂解，平行 亲和层析和凝胶过滤层析。结晶设施目前具有达到峰值的能力 每天准备3000个结晶实验，足够每天筛选8个蛋白质。有些很高 价值目标的生产数量不足以进行标准处理，将对其进行筛选 在UCB纳米结晶设施进行结晶。UCB纳米结晶设施已经开发出 每年使用Fluidigm筛选多达100种蛋白质的384种条件的基础设施 筛选芯片。筛选芯片每96次实验需要2uL的蛋白质。X光设备有2个 每个月在高级光源(ALS)预先计划的光束时间天数。这段时间足以 筛选晶体并收集MAD数据，以确定每年多达30-40个从头开始的结构。这个 本建议书中描述的核心实验室对于成功完成以下总体目标至关重要 计划项目。

The Cloning, Protein Production, Crystallization, and X-ray Data Collection Core constitute a distributed pipeline for X-ray crystal structure determination. The distributed structure takes advantage of local expertise, with the cloning facility at Texas A&M University, the protein purification facility at Los Alamos National Labs, the crystallization facility at Lawrence Livermore National Laboratory, the nanocrystallization facility at UC Berkeley and the X-ray data collection and analysis core located at the Advanced Light Source. This core group is responsible for the majority of the cloning, purification, crystallization and data collection with the program project. The overall goal of this group of core laboratories is to: 1) provide a rapid access library of expressible clones for structural, biochemical and genetic studies, 2) provide highly purified target proteins with sufficient quantity for structure determination through standardization, automation, and parallelization of protocols 3) conduct crystal screens for crystal studies 4) provide screening solutions for inhibitor screens, 5) collect high-resolution MAD data to solve 30-40 structures per year. The pipeline facilities are capable of conducting large-scale clone preparation using a modified Gateway cloning system (Invitrogen) that incorporates a cleavable fusion tag into the system and allows for expression in both E. coli and M. smegmatis. The protein production facility has the capacity to produce 6-8 highly purified target proteins per week using high yield auto-inducing fermentation, parallel cell lysis, parallel affinity and gel filtration chromatography. The crystallization facility currently has the peak capability to prepare 3000 crystallization experiments daily, which is sufficient to screen 8 proteins per day. Some high value targets cannot be produced in quantities sufficient for standard processing and will be screened for crystallization at the UCB nanocrystallization facility. The UCB nano-crystallization facility has developed the infrastructure to screen 384 conditions each for as many as 100 proteins annually using the Fluidigm screening chips. The screening chip requires <2uL of protein per 96 experiments. The X-ray facility has 2 days per month pre-scheduled beam time at the Advanced Light Source (ALS). This time is sufficient to screen crystals and collect MAD data for the determination of up to 30-40 de novo structures per year. The core labs described in this proposal are essential for the successful completion of the overall goals of this program project.