NOVEL VERTEBRATE PROTEIN PRODUCTION--HIV

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.