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

Rayalam S., Eizenstat L.D., Hoenig M., et al.

Conference Proceedings, (2005). American College of Veterinary Internal Medicine, Baltimore: 


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