Protein Production Core

项目总结(蛋白质生产核心) 蛋白质生产核心(PPC)是俄克拉荷马眼镜蛇在结构生物学方面的研究支持 (OCSB)。它是一种最先进的OCSB支持蛋白的表达和纯化设备 研究人员和更广泛的结构生物界。PPC以可靠的方式生产高质量的蛋白质 以收费和高效的方式提供所有方面的实践培训和专家意见 可在该设施中进行蛋白质表达和纯化。PPC工作人员由一名主任菲利普博士组成 伯恩和一个研究生培训职位。研究生通常在PPC中协助一年 这学期。他们接受了PPC主任的培训，使用设施中的所有设备，并在白天协助- PPC今天的活动。PPC由一个由一名初级人员和两名初级人员组成的咨询委员会指导 资深教研员。购买力平价机制是利用OCSB第一阶段资金建立的。里程碑 在此期间取得的成就包括：(1)翻新实验室空间，建立一个独立的安全核心 设施，(2)建立具有现代蛋白质表达和纯化仪器的设施，以及(3) 开发用户基础。用户群已经增长到22个研究小组，其中包括超过65个 个人PPC用户。用户基础生产率提高的证据证明了这一进展 即出版物和赠款支助。 PPC与研究人员和OCSB支持的大分子结晶学密切合作 实验室(MCL)为结晶试验生产均一、高纯度的蛋白质。购买力平价是 配备有个别研究实验室经常找不到的仪器和专业知识。 PPC内的设备包括两个冷藏中压层析系统和一个 针对不同纯化技术(亲和性、疏水性、离子交换性和大小)的大量柱选择 排除)。其他核心设备包括：Emulsiflex-C3均质机，用于裂解酵母和细菌 细胞；一台高性能Avanti离心机、两台冰柜(-80°C和-20°C)和一台BioRad ChemiDoc成像仪 它可以检测紫外线、白色和化学发光。 在拟议的OCSB第二阶段期间，计划的购买力平价扩展包括增加一个低成本 压力层析系统，以减轻两个中压系统的负担，并扩大 该设施的蛋白质提纯吞吐量。增加一个小的自含式动态光散射 仪器将允许在结晶试验之前评估样品的质量。这项补充是理想的 多用户设施，在结晶实验之前对样品质量进行快速评估 非常有用。蛋白质生产能力的提高和生物物理特性的扩展 将满足现有需求，并将有助于扩大用户基础。更大的用户基础将导致更多 来自使用费的收入，这将使设施更接近自给自足。

PROJECT SUMMARY (Protein Production Core) The Protein Production Core (PPC) serves as research support for the Oklahoma COBRE in Structural Biology (OCSB). It is a state-of the art facility for the expression and purification of protein for OCSB-supported researchers and the wider structural biology community. The PPC produces high quality proteins in a reliable and efficient manner on a fee-for-service basis and provides hands-on-training and expert advice in all aspects of protein expression and purification available in the facility. The PPC staff consists of a Director, Dr. Philip Bourne and a Graduate Student training position. The graduate student typically assists in the PPC for a semester. They are trained by the PPC director to use all of the equipment in the facility and assist in the day- to-day activities of the PPC. The PPC is guided by an advisory committee comprised of one junior and two senior faculty researchers. The PPC facility was established using OCSB Phase I funding. Milestones achieved during this period included: (i) renovation of laboratory space to create a stand-alone secure core facility, (ii) establishing the facility with modern instrumentation for protein expression and purification, and (iii) development of a user base. The user base has grown to twenty two research groups, including over 65 individual PPC users. Progress has been demonstrated by evidence of increased productivity of the user base i.e., publications and grant support. The PPC works closely with the investigators and the OCSB-supported Macromolecular Crystallography Laboratory (MCL) to produce homogeneous, highly purified protein for crystallization trials. The PPC is equipped with instrumentation and expertise frequently not found within individual research laboratories. Equipment housed in the PPC includes two refrigerated medium pressure chromatography systems and a large selection of columns for different purification techniques (affinity, hydrophobic, ion-exchange, and size exclusion). Other core equipment includes: an Emulsiflex-C3 homogenizer for lysis of yeast and bacterial cells; a high performance Avanti centrifuge, two freezers (-80 °C and -20 °C), and a BioRad ChemiDoc imager that detects UV, white and chemiluminescence. During the proposed Phase II of the OCSB, planned expansion of the PPC includes the addition of a low pressure chromatography system to ease the burden on the two medium pressure systems and expand the protein purification throughput of the facility. The addition of a small self-contained Dynamic Light Scattering instrument will allow for assessing the quality of samples prior to crystallization trials. This addition is ideal for a multi-user facility where a quick assessment of sample quality prior to crystallization experiments would be extremely useful. The increased protein production capacity and the expanded biophysical characterization will meet the existing demand and will help expand the user base. The larger user base will lead to more income from user fees and this will move the facility closer to self-sufficiency.