Core--Protein Purification

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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.