Protein Technology Core

蛋白质技术核心

• 批准号: 10703087
• 负责人: Anna Valkov
• 金额: $ 140.91万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10703087
• 关键词: Affinity; Antibodies; Biochemical; CCR; Cloning; Collaborations; Cryoelectron Microscopy; Custom; Development; Directed Molecular Evolution; Engineering; Ensure; Enzymes; Equipment; Equipment and supply inventories; Generations; Goals; Human Cell Line; Human Resources; Ice; Image; Individual; Institutional Policy; Intellectual Property; Laboratories; Laboratory Research; Libraries; Mass Spectrum Analysis; Membrane Proteins; Methodology; Microfluidics; Modernization; Modification; Molecular Biology; Multiprotein Complexes; Preparation; Production; Productivity; Protein Engineering; Proteins; Protocols documentation; Reagent; Records; Research; Research Personnel; Research Support; Resolution; Resources; Site; Streptavidin; Structure; Technology; Training; Training Support; Use of New Techniques; Work; base; biophysical properties; crosslink; design and construction; digital; experience; improved; interest; milligram; nanobodies; nanodisk; operation; particle; programs; protein complex; protein protein interaction; protein purification; protein structure function; protocol development; scaffold; skills; structural biology; tissue culture; tool

The Protein Purification and Protein Engineering Cores supported and critically contributed to the research programs of several laboratories at NCI's Center for Structural Biology from October 2021 to June 2022. Specifically, these efforts centered on providing biochemical protein reagents such as site-specific proteolytic and glycolytic enzymes for the cleavage of covalently fused protein tags during the purification of proteins of interest. In additional efforts, the staff produced proteins such as engineered streptavidin and nanobodies for the preparation of customized affinity matrices as well as enzymes for molecular biology applications. The work of research laboratories focused on membrane proteins was supported by the Cores through regular production of high-quality proteins required for the generation of nanodisc assemblies. The Cores also contributed to individual research programs supporting the work of postdoctoral and postbaccalaureate fellows by establishing protocols for the production of their target proteins. Core staff also assisted researchers with tissue culture by maintaining stocks of human cell lines to produce challenging proteins and multiprotein complexes. The Protein Technologies Core (PTC) was established and started its operations as a replacement for the Protein Purification and Protein Engineering Cores on July 1, 2022. The PTC will incorporate and expand the protein production capabilities by retaining experienced personnel who are all highly experienced in protein production, purification, biophysical characterization, and tissue culture. An immediate priority for the PTC is to provide the personnel with additional training and professional development to modernize and refresh their skills. The equipment for protein purification is undergoing significant modernization with the latest state-of-the-art platforms, including microfluidic capabilities already procured for the facility. The production of proteins is an essential requisite for structural biology applications. However, the ability to produce milligrams of pure and biochemically stable proteins and multiprotein complexes can pose significant challenges for individual labs. The PTC will aim to support researchers in this task by employing semi-automated, multi-pronged approaches at each workflow stage, from construct design to cloning, expression, and purification, through the characterization of most protein targets, including membrane proteins and multi-protein complexes. At the outset, the PTC will implement a digital lab notebook to trace and visualize the exact lab workflow. Digital record management will significantly improve productivity and protocol development through collaboration between the PTC staff and research labs. In addition, digitization of PTC records will ensure compliance with the storage and inventory of valuable new biochemical reagents to safeguard valuable intellectual property and ensure compliance with institutional policies. An early and essential strategic goal for the PTC is to establish a pipeline for producing and characterizing binder proteins. Binders are a broad characterization applied to proteins such as single-chain camelid antibodies (nanobodies) and their derivatives that interact with high affinity with a chosen target protein. Binders have broad applications in structural biology as tools for stabilizing and purifying proteins and protein complexes. One important application is in single-particle cryoelectron microscopy of membrane proteins, where binders help find favorable orientations of particles in vitreous ice to enable structure determination. The PTC will establish the cloning, production, purification, and characterization of binders to several important targets from libraries derived from the directed evolution of nanobody scaffolds. The application of binders developed and discovered by the PTC will be further extended to super-resolution imaging and crosslinking mass spectrometry to study protein structure, functions, and protein-protein interactions. In addition to technology and research support, the PTC will be committed to providing training and support for researchers at all levels with particular needs with protein production and characterization.

The Protein Purification and Protein Engineering Cores supported and critically contributed to the research programs of several laboratories at NCI's Center for Structural Biology from October 2021 to June 2022. Specifically, these efforts centered on providing biochemical protein reagents such as site-specific proteolytic and glycolytic enzymes for the cleavage of covalently fused protein tags during the purification of proteins of 兴趣。在额外的努力中，工作人员生产的蛋白质，例如工程链霉亲和蛋白和纳米型，用于制备定制的亲和力矩阵以及用于分子生物学应用的酶。核心通过定期生产纳米盘组件所需的高质量蛋白质来支持，这些研究实验室的工作得到了膜蛋白的支持。这些核心还为个人的研究计划做出了贡献，该计划通过建立生产目标蛋白的协议来支持博士后和后核后研究员的工作。核心人员还通过维护人类细胞系的库存来产生具有挑战性的蛋白质和多蛋白络合物，从而协助研究人员进行组织培养。建立了蛋白质技术核心（PTC），并于2022年7月1日开始运营，以替代蛋白质纯化和蛋白质工程核心。PTC将通过保留在蛋白质生产，纯化，生物物理特征和组织培养中经验丰富的经验丰富的经验丰富的经验丰富的经验丰富的人员，从而结合和扩展蛋白质生产能力。 PTC的直接优先事项是为人员提供额外的培训和专业发展，以现代化和刷新他们的技能。蛋白质净化的设备正在通过最新的最新平台进行重大现代化，包括已经为该设施采购的微流体功能。蛋白质的产生是结构生物学应用的必要必要条件。但是，生产纯和生物化学稳定蛋白和多蛋白络合物的毫克的能力会对单个实验室构成重大挑战。 PTC将通过在每个工作流程阶段采用半自动的多管齐下方法来支持该任务中的研究人员，从构造设计到克隆，表达和纯化，通过表征大多数蛋白质靶标，包括膜蛋白和多蛋白质络合物。首先，PTC将实现数字实验室笔记本，以跟踪和可视化确切的实验室工作流程。数字记录管理将通过PTC员工和研究实验室之间的协作大大提高生产率和协议的发展。此外，PTC记录的数字化将确保遵守有价值的新生化试剂的存储和清单，以保护有价值的知识产权并确保遵守机构政策。 PTC的早期和基本战略目标是建立生产和表征粘合剂蛋白的管道。粘合剂是一种广泛的表征，应用于蛋白质，例如单链骆驼抗体（纳米抗体）及其与所选靶蛋白相互作用的衍生物。粘合剂在结构生物学中具有广泛的应用，作为稳定和纯化蛋白质和蛋白质复合物的工具。一种重要的应用是在膜蛋白的单粒子冷冻电子显微镜中，其中粘合剂有助于找到玻璃体冰中颗粒的有利方向以实现结构测定。 PTC将建立将粘合剂的克隆，生产，纯化和表征化为从纳米机支架的定向演化中得出的几个重要目标的克隆，生产，纯化和表征。 PTC开发和发现的粘合剂的应用将进一步扩展到超分辨率成像和交联质谱，以研究蛋白质结构，功能和蛋白质 - 蛋白质相互作用。除了技术和研究支持外，PTC还将致力于为具有蛋白质生产和表征的各个级别的研究人员提供培训和支持。