A 17 kilobase pair fragment of DNA containing the human TSH (hTSH) β-subunit gene was isolated from a human leukocyte genomic library. Using a 621 base pair human CG α-subunit cDNA and a 2.0 kilobase pair genomic fragment of hTSHβ containing both coding exons, we constructed hCGα and hTSHβ expression vectors containing either the early promoter of simian virus 40 or the promoters of adeno-associated virus. Cotransfection of two adeno-as-sociated virus vectors, each containing one subunit of hTSH, together with a plasmid containing the adenovirus VA RNA genes produced hTSH as well as free human α- and TSHβ-subunits in an adenovirus transformed human embryonal kidney cell line (293). The levels of protein expression in this system were 10- to 100-fold greater than that found in a simian virus transformed monkey kidney cell line (COS) using vectors containing the early promoter of simian virus 40. The hTSH synthesized in 293 cells was glycosylated as indicated by complete binding to concanavalin A-Sepharose but was larger in apparent molecular weight than a standard hTSH preparation on gel chromatography suggesting an altered glycosylation pattern. However, it was immunologically and biologically indistinguishable from two pituitary hTSH standards in an immunora-diometric and in vitro iodide trapping assay, respectively.
ASJC Scopus subject areas
- Molecular Biology