P-205: Production of Recombinant Fish FSH Hormone in Pichia Pastoris

Background: Follicle-stimulating hormone (FSH) belongs to the family of glycoprotein hormones that composing alpha and beta subunits with non-covalently bonds. This hormone involve in regulation of the reproductive processes such as gamete generation and follicular growth. Injection of the hormone in most of fish species increases 17 beta-estradiol production by ovarian tissue and also stimulates 11-keto testosterone production in testes. The purpose of this research is production of recombinant fish FSH hormone (rfFSH) by Pichia pastoris expression system under post-translational modification. Materials and Methods: The alpha and beta fish FSH chains was separately cloned in pTZ57R / T vector and was subcloned into pHILS1 expression vector. This vector (pHILS1) was transformed into competent cell (Pichia pastoris yeast) by electroporation. Transformed clones were suspended in BMMY liquid medium and induced by 0.5% methanol to high levels of expression. The medium was centrifuged at 14000 rpm for 4 minutes for separating Yeast cell from media. Finally, the separated media containing target protein concentrated by Amicon Ultra centrifugal filter for detecting protein by SDS-PAGE and Western blotting techniques. Results: The integration of both vector including alpha and beta FSH genes in the yeast genome was confirmed by PCR with pHILS1 vector-specific primers (5'AOXI and 3'AOXI). Also expression and secretion of rfFSH hormone was confirmed by SDS-PAGE and Western blotting techniques. Conclusion: Pichia pastoris can be an ideal protein expression system for producing a protein with high specific biological activity and correct folding pattern, also it can be used as a model for increasing expression level of glycoprotein hormones by recombinant DNA technology.