Engineering a Bicistronic Expression System for Mammalian Cell-Based Production of Biofunctional Recombinant Follicle Stimulating Hormone (FSH) of Bos Indicus Origin.

Assisted reproductive technologies have become crucial in addressing the growing global demand for livestock production. Follicle-stimulating hormone (FSH) is essential for superovulation in ART procedures. Animal pituitary-derived FSH is widely utilized for these purposes; however, it is costly and presents other limitations. Currently, there are very few commercially available recombinant bovine FSH products, primarily due to challenges in equimolar expression of the FSHα and FSHβ subunits, inefficient post-translational modifications that result in improper heterodimerization, and loss of bioactivity. This study, for the first time, reports the isolation and cloning of FSHα and FSHβ cDNA from Indian cattle (Bos indicus) into a bicistronic-mammalian vector, co-expressed using the P2A peptide. This approach facilitates simultaneous expression and proper dimerization in an in vitro system, producing bioactive recombinant FSH. Additionally, we identified a single-nucleotide-polymorphism (SNP) in the FSHβ region, resulting in a Serine-to-Glycine substitution, unique to Bos indicus. Notably, this SNP was found to be conserved across Indian cattle breeds. This study successfully demonstrates, for the first time, the efficient in vitro production of FSH, thereby overcoming the challenges associated with large-scale commercial production. This advancement will aid in the production of indigenous recombinant bovine FSH, enhancing outcomes in cattle breeding.