LARGE-SCALE SYNTHESIS OF RECOMBINANT HUMAN THYROTROPIN USING METHOTREXATE AMPLIFICATION - CHROMATOGRAPHIC, IMMUNOLOGICAL, AND BIOLOGICAL CHARACTERIZATION

Studies of human TSH (hTSH) structure and function have been limited b y difficulties in producing large quantities of recombinant hormone. W e describe a system for the stable expression of high levels of recomb inant human TSH (rec hTSH) using a mutant form of dihydrofolate reduct ase (dhfr) as an amplifiable dominant selectable marker. A vector expr essing both the hTSH alpha-subunit and the mutant dhfr was cotransfect ed with a hTSH beta-subunit expression vector into dhfr-deficient cell s. Amplification of the transfected sequences by methotrexate selectio n, followed by cell culture in a hollow fiber perfusion system, yielde d rec hTSH production as high as 100,000 mu U/mt. Immunoradiometric as says using five different antibodies revealed no differences in the im munological 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 lucif erase reporter gene. The ED(50) for bovine TSH in this bioassay was 1. 4 ng/mL (3.5 x 10(-11) mol/L). The ratio of the ED(50) 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 tech niques for the high level production of rec hTSH that is immunological ly and biologically equivalent to pituitary hTSH. The ability to produ ce large quantities of rec hTSH using standard laboratory techniques s hould facilitate future studies, such as the development of clinically useful TSH analogs.