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 PEAKâ„¢ 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 Î¼g/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 125I-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 (Emax) suggesting the possibility of the recombinant glycohormone constructs acting as partial agonists at the human TSH receptor. Â© 2005 Elsevier Inc. All rights reserved.
Domestic animal endocrinology
Rayalam, Srujana; Eizenstat, L. D.; Davis, R. R.; Hoenig, M.; and Ferguson, D. C., "Expression and purification of feline thyrotropin (fTSH): Immunological detection and bioactivity of heterodimeric and yoked glycoproteins" (2006). PCOM Scholarly Papers. 646.
This article was published in Domestic animal endocrinology, Volume 30, Issue 3, Pages 185-202.The published version is available at http://dx.doi.org/10.1016/j.domaniend.2005.07.004.
Copyright © 2006 Elsevier.