title: Expression and purification of feline thyrotropin (fTSH): immunological detection and bioactivity of heterodimeric and yoked glycoproteins.
abstract: 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.
authors.affiliation: Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.
authors.forename: S
authors.initials: S
authors.lastname: Rayalam
authors.forename: L D
authors.initials: LD
authors.lastname: Eizenstat
authors.forename: R R
authors.initials: RR
authors.lastname: Davis
authors.forename: M
authors.initials: M
authors.lastname: Hoenig
authors.forename: D C
authors.initials: DC
authors.lastname: Ferguson
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western / Q000662:veterinary; D016466:CHO Cells; D002415:Cats / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000662:veterinary; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D015292:Glycoprotein Hormones, alpha Subunit / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007118:Immunoassay / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D013972:Thyrotropin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D014162:Transfection
chemical_list: D015292:Glycoprotein Hormones, alpha Subunit; D011994:Recombinant Proteins; D013972:Thyrotropin; D000262:Adenylyl Cyclases