Core--Protein Purification

蛋白质纯化核心是一个共享设施，提供必要数量的纯化的、具有生物活性的丙二醛-7蛋白质，用于在本提案的各种项目中提出的假设驱动的研究中用作关键试剂。利用蛋白质治疗前列腺癌(项目1，Paul B.Fisher)、恶性胶质瘤(项目2，Paul Dent)和卵巢癌(项目3，David T.Curiel)将基于先前的数据进行研究，这些数据表明，在细菌中表达的丙二醛-7的GST融合(GST-MDA-7)显示出与感染Ad.mda-7后相同的癌细胞特异性凋亡诱导活性。除了包括未融合GST的控制蛋白外，该核心最初还将为这些项目中的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，该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规模的MDA-7蛋白。该蛋白将在项目1、2和3中提出的基于动物模型的肿瘤灌注研究中具有特定的用途。核心还将能够产生额外的表达载体系统和生产纯化的蛋白，以满足PPG组件项目的特定试剂需求。这些可能包括体外试验中使用的RAS和MAP激酶通路的特定纯化成分，以激活可能由MDA-7触发的特定类型的A信号转导级联反应。将这些活动作为一种共享资源集中起来，将使这些试剂能够通过利用这一领域的现有专门知识，以高效和具有成本效益的方式生产。

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.