Cloning and Sequencing of Feline Thyrotropin (fTSH): Heterodimeric and Yoked Constructs.

Rayalam S, Eizenstat LD, Hoenig M, et al.

in Scientific Proceedings (Abstract). American College of Veterinary Internal Medicine 2005.

Hyperthyroidism is the most common endocrine disorder of elderly cats. However, its early diagnosis and studies of risk factors have been hampered by the inavailability of a feline-specific TSH assay. Although there have been attempts to use available canine TSH immunoassays in cat sera, cross-reactivity is insufficient for distinguishing suppressed values from normal. Feline-specific peptide and antibody reagents are critical for development of a clinically useful immunoassay. In the current study, the genes encoding the mature common pituitary alpha and hormone-specific beta subunits of feline thyroid stimulating hormone (fTSH) were cloned and sequenced. The feline common pituitary alpha gene was cloned from the total 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 the feline genomic DNA by direct polymerase chain reaction (PCR). Following the experience with improved expression of the human TSH beta subunit, the second intron was included to produce a fTSHbeta mini-gene construct. For both subunits, primers were based on consensus sequences from TSH in other species. The resulting 510 bp PCR product for the alpha subunit included the full coding sequence of the 96 amino acid mature subunit preceded by that of a 24 amino acid signal peptide. The predicted amino acid sequence of the mature α subunit had the following species homologies: to canine (98%), bovine (95%), tiger (97%) and human (69%). 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 mature fTSHbeta subunit is homologous to canine (94%), human (88%), bovine (91%) and equine (95%) TSHbeta subunits. An immunoaffinity tag FLAG was added to 3’ end of the alpha gene to facilitate detection by Western blot and purification. In human pituitary glycoproteins, single chain or yoked analogues have been shown to have increased stability and bioactivity. In the current study, yoked fTSH (yfTSH) was developed by fusing the nucleotides encoding the C-terminus of the beta subunit to the N-terminus of the alpha subunit by using DNA encoding the C-terminal peptide (CTP) of human chorionic gonadotropin beta subunit of 26 amino acids as a linker peptide. The yoked fTSH construct encoded from N-terminus to C-terminus: beta-CTP-alpha-FLAG. The construct of 1260 bp was cloned and sequence confirmed. This work describes for the first time the full coding sequences of the two subunits of fTSH and has produced DNA constructs for its in vitro expression and purification.