This is a Phase Il SBIR application pertaining to production of recombinant sheep erythropoietin (Ep). Funding for PI's phase I SBIR proposal generated sheep Ep cDNA. At the same time Dr. Franklin Bunn of Harvard University also cloned sheep Ep cDNA and promises to collaborate with the Applicant in this Phase II endeavor. Dr. Hankin's now proposes to produce purified sheep Ep, characterize biochemical and biological properties of the recombinant protein and conduct in vivo experiments in sheep/human chimera model. First, the Investigator's group will confirm that isolated sheep Ep cDNA has representative sequence by isolating Ep cDNA from at least two additional sheep. They will then test various expression systems for large scale production for sheep Ep. The primary choice is Chinese hamster ovary cells since human Ep is being successfully produced in large quantity using this cell line. Alternative choices for the expression systems are murine HCO cells which he is very familiar with and insect (army worm) cells. Carbohydrate moiety of Ep produced by the insect cells, however, may be different from native sheep Ep. Regarding purification of the recombinant sheep Ep, the Investigators plan to try both classic protein purification procedures as well as affinity procedures. Affinity procedures may be either receptor affinity method in which the murine Ep receptor will be bound to Affi-gel beads or immunoaffinity method in which the monoclonal antibodies prepared against sheep Ep will be bound to the beads. Finally the Investigators will characterize physicochemical and biological properties such as molecular weight, aminoterminal sequence, carbohydrate content of Ep and, reactivity with anti-Ep antibodies. They will also characterize the biological activity of the recombinant Ep in vitro and in vivo methods.
