Cloning, expression and purification of recombinant feline thyrotropin (fTSH)
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The genes encoding the feline common glycoprotein ± (CGA) and feline thyrotropin (fTSH) ² subunits were cloned and sequenced. The feline CGA gene encodes a 96 amino acid peptide and fTSH² sequence encodes a 138 amino acid peptide. A FLAG tag was added to the 3’ end of the CGA gene to facilitate detection and purification. A single chain analogue of fTSH termed yoked fTSH (yfTSH) was developed by fusing C-terminus of the ² subunit using a yoking peptide, CTP to the N-terminus of the ±-subunit. Expression levels of 1 µg/ml were achieved for both TMheterodimeric and yoked fTSH forms in modified HEK293 (PEAK) cells. The glycoproteins were purified in one step using anti-FLAG immunoaffinity column chromatography to high purity. Both heterodimeric and yoked glycoproteins were recognized with 40% detection by both commercial canine TSH immunoassay and an in-house canine TSH ELISA. The heterodimeric and yoked fTSH behaved immunologically parallel with the pituitary - source canine TSH in the in-house ELISA. The heterodimeric and yoked forms of fTSH were 12.5 and 3.4 % as potent as pituitary source bovine TSH at displacing I-bTSH and 45 and 24 % as potent in stimulating adenylate cyclase activity in human TSH receptor-expressing JP09 cells. Also, a reduced maximal effect at maximal concentration (Emax) suggests the possibility of the recombinant peptides acting as partial agonists of the human TSH receptor. For expression of fTSH in baculovirus expression system, the signal peptide in both the subunits was replaced with the honey bee mellitin signal sequence and the intron in the fTSH beta “mini gene” construct was removed with an over-lap PCR. The expression levels as determined by immunoreactivity were 35 ± 15 ng/ml. Since purification of large quantities of recombinant fTSH for standardization by protein assay was not successful, as an alternative, enzymatically desialylated fTSH expressed in TMPEAK cells was prepared and used to characterize the bioactivity. Both the insect cell-expressed and desialylated fTSH behaved immunologically parallel to pituitary-source canine TSH. No significant change in the cAMP production and binding affinity were observed with desialylation. However, the biological to immunological ratio for cAMP production increased significantly with desialylation.