Core--Protein Purification

核心——蛋白质纯化

• 批准号: 7007048
• 负责人: Pankaj GUPTA
• 金额: $ 6.91万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7007048
• 关键词: biomedical facility; chimeric proteins; cytokine; protein purification; tumor suppressor proteins

The Protein Purification Core is a shared facility to provide requisite amounts of purified, biologically active MDA-7 protein for utilization as a key reagent in the hypothesis-driven studies proposed in the various Projects of this proposal. Utilization of protein for therapy of prostate cancer (Project 1, Paul B. Fisher), malignant glioma (Project 2, Paul Dent) and ovarian cancer (Project 3, David T. Curiel) will be studied based on previous data showing that a bacterially expressed GST-fusion of MDA-7 (GST-MDA-7) shows the same cancer cell specific apoptosis inducing activity observed after infection with Ad.mda-7. The core will initially provide required preparative scale (milligram quantities) of purified, biologically tested protein for each of these projects in addition to control protein including unfused GST. In addition, the core will establish by constructing vectors, standardizing and scaling up a procedure based on an insect cell baculovirus expression system, for production of preparative scale MDA-7 protein with a minimal fusion tag. This protein will have specific utility in tumor infusion studies based on animal models, proposed in Projects 1, 2 and 3. The core will also be in a position to generate additional expression vector systems and produce purified protein, to meet specific reagent needs to the component Projects of the PPG. These could include specific purified components of the Ras and MAP kinase pathways used in in vitro tests for activation of specific types of a signal transduction cascades that might be triggered by mda-7. The centralization of these activities as a shared resource will enable these reagents to be produced in an efficient and cost-effective manner by capitalizing on existing expertise in this area.

蛋白质纯化核心是一个共享设施，可提供所需量的纯化的、具有生物活性的 MDA-7 蛋白质，用作本提案各个项目中提出的假设驱动研究中的关键试剂。将根据先前的数据研究利用蛋白质治疗前列腺癌（项目 1，Paul B. Fisher）、恶性神经胶质瘤（项目 2，Paul Dent）和卵巢癌（项目 3，David T. Curiel）表达的 MDA-7 的 GST 融合体 (GST-MDA-7) 显示出与 Ad.mda-7 感染后观察到的相同的癌细胞特异性细胞凋亡诱导活性。除了包括未融合 GST 的对照蛋白外，核心最初将为每个项目提供所需的制备规模（毫克量）的纯化、生物测试蛋白。此外，核心将通过构建载体、标准化和扩大基于昆虫细胞杆状病毒表达系统的程序来建立，用于生产具有最小融合标签的制备级MDA-7蛋白。该蛋白质将在项目 1、2 和 3 中提出的基于动物模型的肿瘤输注研究中具有特定用途。该核心还将能够生成额外的表达载体系统并生产纯化的蛋白质，以满足特定试剂的需求。 PPG 的组成项目。这些可能包括 Ras 和 MAP 激酶途径的特定纯化成分，用于体外测试，以激活可能由 mda-7 触发的特定类型的信号转导级联。这些活动作为共享资源的集中化将使这些试剂能够利用该领域的现有专业知识以高效且具有成本效益的方式生产。