Core--Protein Purification

蛋白质纯化核心是一个共享的设施，用于提供必要数量的纯化的、具有生物活性的MDA-7蛋白，作为本提案中各个项目中提出的假设驱动研究的关键试剂。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）将根据先前的数据进行研究，这些数据表明细菌表达的gst融合MDA-7 （GST-MDA-7）在感染Ad.mda-7后具有相同的癌细胞特异性凋亡诱导活性。除了控制蛋白（包括未融合的GST）外，核心最初将为每个项目提供所需的制备规模（毫克量）纯化的生物测试蛋白。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，以最小的融合标签生产制备规模的MDA-7蛋白。该蛋白将在基于动物模型的肿瘤输注研究中具有特定的效用，在项目1、2和3中提出。该核心还将能够生成额外的表达载体系统并产生纯化蛋白，以满足PPG组成项目的特定试剂需求。这些可能包括在体外试验中使用的Ras和MAP激酶途径的特定纯化组分，用于激活可能由mda-7触发的特定类型的信号转导级联。将这些活动集中起来作为共享资源，将使这些试剂能够利用这一领域的现有专门知识，以有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.