268 BOVINE AND EQUINE RECOMBINANT FOLLICLE-STIMULATING HORMONE PRODUCED IN TWO HETEROLOGOUS HOSTS: ESCHERICHIA COLI AND TRYPANOSOMA BRUCEI

Abstract

Follicle-stimulating hormone (FSH) is a heterodimeric glycoprotein hormone consisting of ± and ² subunits produced by different genes. Following release from the anterior pituitary, FSH is responsible for development of ovarian follicles from the time of emergence of a follicular wave until divergence of the dominant follicle. Treatment of cattle and horses with recombinant FSH would be valuable for induction of superovulation in an effort to harvest multiple embryos for use in embryo transfer. Glycosylation of the subunits may be essential for proper function and receptor recognition, thereby complicating the production of recombinant FSH. Therefore, a eukaryotic, rather than a prokaryotic, host would be most beneficial for generation of recombinant protein. To explore recombinant protein synthesis, we utilized the ancient protozoan Trypanosoma brucei. Perhaps best known for its parasitic life style, T. brucei has been the focus of extensive biochemical and genetic research, with the nonpathogenic insect stage of the parasite serving as a model eukaryote for the study of many fundamental eukaryotic pathways, including glycosyl phosphatidylinositol (GPI) anchor biosynthesis and RNA editing. The ease of molecular manipulation of this protozoan, and the organism's robust glycosylation machinery, has led us to explore the possibility of using the non-pathogenic life stage for recombinant protein production. The objective of this study was to produce recombinant bovine and equine FSH in both prokaryotic and eukaryotic expression systems. Protein has been produced in Escherichia coli using the pQE30 expression system. Subunit genes were ligated into pQE30 and expressed in M15 E. coli cells. These cells were found to produce detectable amounts of the individual subunit protein after a three hour induction. To express recombinant protein in T. brucei, subunit genes were cloned into pLEW2T7(6His), a stably integrated vector designed to allow inducible expression of proteins fused to a C-terminal six-histidine tag. Clones were subsequently transformed into T. brucei and are awaiting protein expression. All protein purification will be performed via nickel affinity chromatography and protein concentration will be measured using radioimmunoassay. Bioactivity will be analyzed using a rat ovarian weight assay prior to use in cattle and horses. The purified protein will be administered as intramuscular injections twice daily for four days beginning on the day of follicular wave emergence. Producing recombinant FSH will be useful for the embryo transfer and food safety industries by increasing consistency of biologic activity, decreasing production costs, and, perhaps most importantly, decreasing the opportunity for zoonotic transmission of prions such as bovine spongiform encephalopathy.