Purification and Characterization of a Recombinant Glycoprotein, Canine Thyroid Stimulating Hormone, as a Prelude to the Development of the Reproductive Glycoproteins
Delovio, Malcolm Leihulu
MetadataShow full item record
A baculovirus (Spodoptera frugiperda) system was designed to express recombinant canine thyroid stimulating hormone (rcTSH). The efficacy of rcTSH was measured against pituitary derived bovine thyroid stimulating hormone (bTSH) through a series of in vitro and in vivo experiments. Initial purification of rcTSH was performed in order to characterize the hormone for further analyses. Ion exchange columns and tangential flow membranes were chosen based upon the traits of the rcTSH molecule. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels visualized by Coomassie blue, silver stain, and western blot demonstrated the effectiveness of the purification process. Primary cell, static tissue, and perifusion tissue cultures were employed to investigate in vitro thyroid cell/tissue response to rcTSH and bTSH. Canine thyroid cells were liberated from tissue samples, cultured, then exposed to TSH treatments in which media was subsequently harvested and measured for cyclic adenosine monophosphate (cAMP), a second messenger in the TSH downstream signaling cascade. The cAMP concentrations measured were sporadic and not consistent with expected concentrations for treatments or controls. For the static tissue culture, slices of bovine thyroid tissue were incubated and exposed to a series of media-only wash steps as well as treatment steps using varying concentrations of rcTSH. Unfortunately, the experiment was compromised resulting in the slow release of thyroxine (T4) for all samples due to tissue death. Perifusion experiments conducted on bovine thyroid tissue compared the release of T4 due to bTSH and rcTSH stimulation in a dynamic system. Unable to perform statistical analysis due to small sample sizes, graphical representation demonstrated stimulatory effects by bTSH and rcTSH when compared to control. Biological assays were used to compare the in vivo efficacy of rcTSH to bTSH which included 3 species (goldfish, rat, and canine). Results from mammalian experiments when subjected to analysis of variance (ANOVA) resulted in the rejection of the null hypothesis of equal means (P<0.05) between control, bTSH, and rcTSH treatments. Further analysis by Tukey's W procedure demonstrated the stimulatory actions of rcTSH and bTSH to be similar (P>0.05) to each other while greater (P<0.05) than control at the 4 and 6 hour post injection time.