title: Large scale synthesis of recombinant human thyrotropin using methotrexate amplification: chromatographic, immunological, and biological characterization.
abstract: Studies of human TSH (hTSH) structure and function have been limited by difficulties in producing large quantities of recombinant hormone. We describe a system for the stable expression of high levels of recombinant human TSH (rec hTSH) using a mutant form of dihydrofolate reductase (dhfr) as an amplifiable dominant selectable marker. A vector expressing both the hTSH alpha-subunit and the mutant dhfr was cotransfected with a hTSH beta-subunit expression vector into dhfr-deficient cells. Amplification of the transfected sequences by methotrexate selection, followed by cell culture in a hollow fiber perfusion system, yielded rec hTSH production as high as 100,000 microU/ mL. Immunoradiometric assays using five different antibodies revealed no differences in the immunological activities of rec hTSH and pituitary hTSH. Bioactivity was measured in a novel TSH bioassay coupling the generation of cAMP by a transfected hTSH receptor to the cAMP-dependent regulation of a luciferase reporter gene. The ED50 for bovine TSH in this bioassay was 1.4 ng/mL (3.5 x 10(-11) mol/L). The ratio of the ED50 values for rec hTSH and pituitary hTSH was 1.0:1.1 (P = NS), indicating that the two TSHs were of equivalent potency. In conclusion, we have developed techniques for the high level production of rec hTSH that is immunologically and biologically equivalent to pituitary hTSH. The ability to produce large quantities of rec hTSH using standard laboratory techniques should facilitate future studies, such as the development of clinically useful TSH analogs.
authors.affiliation: Department of Medicine, University of Maryland School of Medicine, Baltimore, USA.
authors.forename: A
authors.initials: A
authors.lastname: Hussain
authors.forename: C A
authors.initials: CA
authors.lastname: Zimmerman
authors.forename: J A
authors.initials: JA
authors.lastname: Boose
authors.forename: J
authors.initials: J
authors.lastname: Froehlich
authors.forename: A
authors.initials: A
authors.lastname: Richardson
authors.forename: R S
authors.initials: RS
authors.lastname: Horowitz
authors.forename: M T
authors.initials: MT
authors.lastname: Collins
authors.forename: R W
authors.initials: RW
authors.lastname: Lash
mesh_terms: D000818:Animals; D001681:Biological Assay; D016466:CHO Cells / Q000378:metabolism; D002417:Cattle; D002845:Chromatography; D006224:Cricetinae; D006801:Humans; D007158:Immunologic Techniques; D008727:Methotrexate / Q000494:pharmacology*; D011994:Recombinant Proteins; D013972:Thyrotropin / Q000096:biosynthesis*; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D013972:Thyrotropin; D008727:Methotrexate