Open AccessCloning of the Human Thyrotropin β-Subunit Gene and Transient Expression of Biologically Active Human Thyrotropin after Gene Transfection
Author(s) -
Fredric E. Wondisford,
Stephen J. Usala,
G. Stephen DeCherney,
Maija L. Castrén,
Sally Radovick,
Peter W. Gyves,
James P. Trempe,
B P Kerfoot,
Vera M. Nikodem,
B J Carter
Publication year - 1988
Publication title -
molecular endocrinology
Language(s) - English
Resource type - Journals
eISSN - 1944-9917
pISSN - 0888-8809
DOI - 10.1210/mend-2-1-32
Subject(s) - biology , microbiology and biotechnology , transfection , complementary dna , gene , gene expression , reporter gene , expression vector , virology , recombinant dna , biochemistry
A 17 kilobase pair fragment of DNA containing the human TSH (hTSH) beta-subunit gene was isolated from a human leukocyte genomic library. Using a 621 base pair human CG alpha-subunit cDNA and a 2.0 kilobase pair genomic fragment of hTSH beta containing both coding exons, we constructed hCG alpha and hTSH beta expression vectors containing either the early promoter of simian virus 40 or the promoters of adeno-associated virus. Cotransfection of two adeno-associated 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 alpha- and TSH beta-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 immunoradiometric and in vitro iodide trapping assay, respectively.