Production of a high performance recombinant yeast secreting equine follicle stimulating hormone

During each estrous cycle, mares release only one mature and fertile ovule. In each cycle they have more than one follicle are under control of follicle stimulating hormone (FSH), which all of them possess the potential of maturation but just one of them can grow and release the ovule. This is because the FSH released from the pituitary gland is inhibited by the first released egg. Therefore, enough exogenous FSH can be used for ovary stimulation or superovulation. In this study we aimed to produce equine FSH in a much more cost-effective and faster growth host than production in mammalian cell lines. For this purpose the coding sequence of equine FSH gene was obtained from NCBI data bank, then appropriate yeast secretion signal peptide and suitable linker between its two subunits were added to the sequence and subsequently codon-optimized for better expression in the yeast host. The optimized sequence was then synthesized in the pUC57 vector. After PCR amplification of a fragment containing the recombinant FSH sequence, the construct was cloned to p316TDH3 yeast vector using appropriate restriction enzymes, for both construct and vector, and T4 DNA ligase. The correct cloned vectors were identified by transformation of E. coli (DH5a) and screening by ampicillin selection marker as well as colony PCR and sequencing. After plasmid extraction from positive colonies, the Saccharomyces cerevisiae competent cells, as eukaryotic host for suitable FSH expression, was transfected by p316TDH3-eFSH by using the cold lithium acetate and single-strand DNA carrier solutions. The recombinant yeasts were selected using URA3 auxotrophic marker present within the vector. Our results confirmed that some of the yeast cells have accepted the recombinant p316TDH3 vectors. To sum up, we introduce these recombinant yeasts for cost-effective production of the equine FSH.

Keywords:

equine FSH , eukaryotic host , superovulation , yeast