High level expression of p55, a thyroid hormone binding protein which is homologous to protein disulfide isomerase in a retroviral vector.
To develop an efficient system for a high level expression of a human cellular thyroid hormone binding protein (p55) in eukaryotic cells, a full-length p55 cDNA was inserted into a Harvey murine sarcoma virus-derived vector (pHTBr) and transfected into mouse NIH 3T3 cells. The expressed p55 has a molecular weight of 55,000 and is recognized by the human specific anti-p55 monoclonal antibody. Similar to the endogenous p55, the expressed p55 is localized on endoplasmic reticulum and nuclear envelope. Moreover, p55 was specifically labeled by N-bromoacetyl-3,3',5-triiodo-L-thyronine. Thus, the expressed p55 is structurally indistinguishable from the endogeneous p55. pHTBr was packaged into a virus with the aide of an amphotropic virus. Infection by pHTBr-containing virus yielded a 2-11 fold higher expression than the endogeneous p55 in NIH3T3, rat GH3, human HepG2 cells and a mouse monoclonal antibody secreting hybridoma.
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Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20892.
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Cheng
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D000345:Affinity Labels; D000818:Animals; D001345:Autoradiography; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005822:Genetic Vectors; D006251:Harvey murine sarcoma virus / Q000235:genetics*; D007535:Isomerases / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D019704:Protein Disulfide-Isomerases; D012333:RNA, Messenger / Q000302:isolation & purification; D009053:Sarcoma Viruses, Murine / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013963:Thyroid Hormones; D014162:Transfection; D000097810:Thyroid Hormone-Binding Proteins
D000345:Affinity Labels; D002352:Carrier Proteins; D007535:Isomerases; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D019704:Protein Disulfide-Isomerases; D012333:RNA, Messenger; D013963:Thyroid Hormones; D000097810:Thyroid Hormone-Binding Proteins