P-198: Utilization of Pichia Pastoris Secretion System for Expression of Equine Follicle Stimulating Hormone

Background: Equine follicle stimulating hormone (eFSH) is a pituitary heterodimeric glycoprotein consists of noncovalently linked of generic alpha subunit and a hormone specific beta subunit. The molecular weights of the subunits are similar and about 16 KD. In general, FSH plays a key role in controlling vertebrate gonadal functions. In female mammals, ovarian maturation and follicular growth is critically dependent on FSH stimulation. Materials and Methods: The purpose of this study was to clone and express the Equine follicular stimulating hormone in Pichia Pastoris. Extracted total mRNA from Iranian Thoroughbred horse’s anterior pituitary gland was used to synthesize the appropriate cDNA. The two genes encoding the alpha and beta subunits were cloned after amplification using the two specific primers. The genes were separately cloned into the pTZ57R/T vector before transforming into the DH5α strains of E.coli. Cloning was then confirmed by PCR and sequencing methods following by subcloning into the yeast pic-9 expression vector. The recombinant vectors were linearized by salI and sacI restriction enzymes and transformation into the Pichia Pastoris using the electroporation method. Positive cell lines were grown on Buffered Complex Methanol Medium (BMMY) containing methanol as the sole carbon source. Results: Finally, the expression of recombinant eFSH protein was confirmed by SDS-PAGE and Western blotting methods. Conclusion: Production of recombinant protein in eukaryotic expression systems such as pichia pastoris is a reliable method for producing of therapeutic equine FSH. Also the detection of the recombinant produced protein by specific antibody confirmed a correct post translated modified structure.