Cloning and Expression of Biologically Active Buffalo Thyroid Stimulating Hormone (Butsh) in Methylotropic Yeast Pichiapastoris

Superovulation and Embryo transfer are commonly used techniques for improving genetic potential and disseminating superior germ plasm. The overall results of superovulation and embryo transfer have been discouraging in buffalo. Unfortunately, animals are notably unresponsive to exogenously administered hormones as only heterologous preparations have been used since homologous preparations are not available. Thus, it was of primary importance to develop effective homologous preparations which could be used in field trials. An in vitro assay was standardized to assess the TSH bioactivity. The effect of stimulation of buffalo thyroid follicles by bTSH was measured in the form of released cAMP. Total RNA was extracted from the pituitary and first strand cDNA was made. The cDNA for the specific subunits were amplified using gene specific primers. Heterodimeric buTSH was cloned and expressed in yeast Pichia pastoris. Successful co-expression of the TSH subunits was achieved with an average yield of 5-7 mg/L of the heterodimeric hormone. The recombinant hormone was secreted into the fermentation medium since the hormone subunits were cloned downstream to the yeast Α mating peptide. The recombinant hormone was further purified by hydrophobic interaction chromatography using Phenyl- Sepharose and characterized using similar homologous and heterologous assay systems. The recombinant hormone was active in ex-vivo bioassay and the bioactivity of the purified recombinant buTSH was found to be 8 IU/mg of protein.