Despite major potential advantages, retroviral vectors with cloned genes have not previously been useful for production of large quantities of properly processed vertebrate proteins. Recently, we developed an approach that can solve this problem, and we have thereby isolated cell lines that produce secretory human growth hormone (hGH) as 4% of total protein (c.a., 6 mg/liter - 24h). We have also expressed genes for a membrane glycoprotein and for HIV protease-reverse transcriptase. I now propose to thoroughly analyze this protein production method, to increase its yields, and to apply the improved process to production of human erythropoietin and HIV proteins for structural and functional investigations. When retroviral vector DNAs are transfected into cocultures of an esotropic packaging cell and an amphotropic packaging cell, they become spontaneously amplified to high proviral multiplicities (copy numbers per cell) by an infectious process in the absence of transmissible helper virus. Because cells resist infection by retroviruses that have envelope glycoproteins of the same host- range type they synthesize, helper-free virions from either packaging cell can only infect the other. This results in theoretically limitless back-and-forth (""""""""ping-pong"""""""") amplification. Using the gene for hGH as a model, we will (1) Thoroughly analyze the method and improve protein yields. (2) Improve our ping-pong vector by replacing its enhancer-promotor with one that we have demonstrated to be ten times more active in fibroblasts. Then we will (3) Clone the human erythropoietin gene and express it maximally. Purify the Epo and study its structure and function. Use the byproduct virions as an autocrine model for murine erythroleukemia. (4) Produce large quantities of specific native HIV proteins including env glycoproteins and tat. (5) Use the high high titer byproduct virions to study expression of these proteins in different cells. (6) Purify HIV proteins for structural and functional investigations. Structural investigations will include crystallization and X-ray diffraction endeavors. These studies will thoroughly characterize a novel and promising method for producing vertebrate proteins, and will also result in important information about leukemia and AIDS.
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