Overexpression of an immunologically-intact, secreted form of human thyroid peroxidase in eukaryotic cells

• Published in: Molecular and cellular endocrinology Vol. 78; no. 1; pp. 107 - 114
• Main Authors: D. Kaufman, Keith, Foti, Daniela, Seto, Pui, Rapoport, Basil
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier Ireland Ltd 01.06.1991; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Biological and medical sciences; Chinese hamster ovary cell; CHO Cells - drug effects; CHO Cells - metabolism; Cricetinae; Cricetulus; Dihydrofolate reductase; Enzyme Induction - drug effects; Enzyme-Linked Immunosorbent Assay; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Gene Amplification - drug effects; Genetic Vectors; Humans; Iodide Peroxidase - biosynthesis; Iodide Peroxidase - immunology; Iodide Peroxidase - metabolism; Methotrexate; Methotrexate - pharmacology; Molecular Sequence Data; Oxidoreductases; Plasmids; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - metabolism; Tetrahydrofolate Dehydrogenase - metabolism; Thyroid peroxidase; Transfection; Chinese hamster ovary cell; Dihydrofolate reductase; Thyroid peroxidase; Methotrexate; Human; Enzyme; Rodentia; Thyroid gland; Gene expression; CHO cell line; Vertebrata; Mammalia; Transfection; Blotting assay; Peroxidase; Recombinant protein; ELISA assay; Hamster
• ISSN: 0303-7207; 1872-8057
• DOI: 10.1016/0303-7207(91)90191-T

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.