P-77: Optimization of Ovine FSH Gene Expression in The Pichiapastoris System byRegulating The Culture Conditions

Background: Ovine follicle stimulation hormone (OFSH) is a pituitary glycoprotein and belongs to the family of glycoprotein hormones. This hormone plays a key role in the function of the reproductive system: it is essential for sertoli cell function and spermatogenesis in testis and it stimulates the growth of ovulatory follicles in females. Ovine FSH hormone is a heterodimeric hormone consisting of a 92 amino acid, α subunit, and a 111 amino acid β subunit which are linked non-covalently. Pichia pastoris is a suitable host for the production of foreign proteins and known as the most economical eukaryotic expression system for secretion of heterologous proteins. The purpose of this study is the optimization of ovine FSH gene expression in the pichia pastoris system by regulating the culture conditions consist of temperature and percentages of induction of methanol. Materials and Methods: The expression process was tested in a five-day incubation period in different temperatures (32‚30‚27‚24‚21‚18‚15) and different percentage of induction of methanol (0.5‚1‚2‚3) for three times. After completing this process for each parameter‚ the recombinant proteins were precipitate using TCA method and then protein expression levels were evaluated by SDS-PAGE and then western blotting analysis for these two parameters. Finally, optimum temperature and concentration of induction of methanol during expression process was selected. Results: The experiments were performed in order to find the optimal temperature and methanol induction yield. Results of SDSPAGE and western blotting showed a ~18-kDa band of protein of interest. It is also demonstrated that the best temperature for expression of ovine FSH protein in pichia pastoris system was 300C and the optimal methanol induction yield was 0.5%. Conclusion: The findings demonstrate how temperature and methanol induction influence the OFSH gene expression in the pichia pastoris system.