Overexpression of an immunologically-intact, secreted form of human thyroid peroxidase in eukaryotic cells.
Recombinant human thyroid peroxidase (hTPO) has been expressed in eukaryotic cells as both the membrane-associated enzyme and as a secreted protein. We now report overexpression of the secreted form of recombinant hTPO in eukaryotic cells. For hTPO gene amplification we used a vector containing the mouse dihydrofolate reductase (DHFR) gene. Stably transfected Chinese hamster ovary (CHO) cells were grown in the presence of increasing concentrations of methotrexate (MTX) and hTPO expression was measured immunologically in an enzyme-linked immunosorbent assay (ELISA). Progressive overexpression of secreted hTPO occurred up to a final MTX concentration of 10,000 nM. Slot-blot analysis of genomic DNA from CHO cells expressing truncated hTPO revealed amplification profiles of the DHFR and hTPO genes to be similar, in parallel with hTPO protein production. High-level expression of secreted hTPO offers the potential for obtaining large amounts of biologically and immunologically active protein for future study.
Kaufman
K D
KD
Thyroid Molecular Biology Unit, V.A. Medical Center, San Francisco, CA 94121.
Foti
D
D
Seto
P
P
Rapoport
B
B
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004790:Enzyme Induction / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D005784:Gene Amplification / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D007453:Iodide Peroxidase / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014162:Transfection
D011993:Recombinant Fusion Proteins; D007453:Iodide Peroxidase; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate