Cloning and sequencing of feline thyrotropin (fTSH): heterodimeric and yoked constructs.

The genes encoding the mature common glycoprotein alpha (CGA) and hormone-specific beta subunits of feline thyroid stimulating hormone (fTSH) were cloned and sequenced. The feline CGA gene was cloned from RNA extracted from the feline pituitary gland by the reverse transcription polymerase chain reaction (RT-PCR). The gene fragment that encodes mature TSHbeta was cloned from feline genomic DNA after direct polymerase chain reaction (PCR). In both cases, primers were based on the consensus sequences from TSH in other species. The resulting 510 bp PCR product for the CGA-subunit included the full coding sequence for the 96 amino acid mature subunit preceded by a 24 amino acid signal peptide. The 850 bp sequence of fTSHbeta genomic DNA consisted of two coding exons, an intron of 418 bp, and a 60 bp signal sequence. The octapeptide immunoaffinity tag FLAG was added to 3' end of the alpha gene to facilitate detection and purification. Both genes were cloned independently downstream from the EF1alpha promoter of the PEAK transfer vector to facilitate co-expression studies in PEAK cells (modified human embryonic kidney (HEK) cells). A single-chain analogue of fTSH termed yoked fTSH (yfTSH) was developed by fusing the nucleotides encoding the C-terminus of the beta-subunit fused to the N-terminus of the alpha-subunit with DNA encoding the C-terminal peptide (CTP) of human chorionic gonadotropin beta-subunit as a linker peptide. The resulting single-chain analogue encoded from N-terminus to C-terminus: beta-CTP-alpha-FLAG. The resulting DNA sequence was cloned, sequenced, ligated and recloned into expression vector PEAK. This report constitutes the first cloning and sequencing of the genes encoding the subunits of feline thyrotropin.

Expression and purification of feline thyrotropin (fTSH): immunological detection and bioactivity of heterodimeric and yoked glycoproteins.

The goal of this study was to express and purify recombinant feline TSH as a possible immunoassay standard or pharmaceutical agent. Previously cloned feline common glycoprotein alpha (CGA) and beta subunits were ligated into the mammalian expression vector pEAK10. The feline CGA-FLAG and beta subunits were cloned separately into the pEAK10 expression vector, and transiently co-transfected into PEAK cells. Similarly, previously cloned and sequenced yoked (single chain) fTSH (yfTSH) and the CGA-FLAG sequences were ligated into the same vector, and stable cell lines selected by puromycin resistance. Expression levels of at least 1 microg/ml were achieved for both heterodimeric and yoked fTSH forms. The glycoproteins were purified in one step using anti-FLAG immunoaffinity column chromatography to high purity. The molecular weights of feline CGA-FLAG subunit, beta subunit and yfTSH were 20.4, 17, and 45 kDa, respectively. Both heterodimeric and yoked glycoproteins were recognized with approximately 40% detection by both a commercial canine TSH immunoassay and an in-house canine TSH ELISA. The yoked glycoprotein exhibited parallelism with the heterodimeric form in the in-house ELISA, supporting their possible use as immunoassay standards. In bioactivity assays, the heterodimeric and yoked forms of fTSH were 12.5 and 3.4% as potent as pituitary source bovine TSH at displacing (125)I-bTSH and 45 and 24% as potent in stimulating adenylate cyclase activity in human TSH receptor-expressing JP09 cells. However, in addition to reduced receptor binding affinity, the recombinant glycohormones produced a reduced maximal effect at maximal concentration (E(max)) suggesting the possibility of the recombinant glycohormone constructs acting as partial agonists at the human TSH receptor.