This proposal aims at the development of methods that can be employed to produce large amounts of biologically active thyroid stimulating hormone (TSH). Specifically it will: (1) Obtain the genomic clone that encodes that beta hTSH molecule. Develop convenient restriction digest sites and develop strategies to introduce the clone into an eukaryotic expression vector. (2) Construct the expression vector to insert the alpha cDNA and the genomic beta hTSH into a bovine papilloma virus (BPV) expression casette that contains promoter and poly A sequences. (3) Transfect C127 mouse epithelial cells with the engineered BPV plasmids. (4) Identify transformants by immunological methods. (5) Produce conditioned medium for analysis of secreted hTSH by radioimmunoassay, bioassays, and receptor binding activity.
