High level expression of functional full length human thyroid hormone receptor β1 in insect cells using a recombinant baculovirus

Tomas Barkhem, Bo Carlsson, Jan Simons, Birgitta Möller, Anders Berkenstam, Jan Ake Gustafsson, Stefan Nilsson

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

We have cloned the human thyroid hormone receptor β1 (hThR β) from the human breast cancer cell line T47D using the PCR technique. A recombinant baculovirus transfer vector pVL1392/hThR β was constructed and the full length receptor was expressed in the insect cell line Spodoptera frugiperda (Sf9). Approx. 10-15 × 106 receptors are expressed/ cell which implies a production level of 2.5-4.0 mg hThR β/1 of cell culture. The expressed hThR β displayed a single class of binding sites for T3 with high affinity. Western blot analysis using a polyclonal antibody indicated that the molecular weight of the baculovirus expressed receptor is approx. 50 kDa. Crude nuclear extract of hThR β labeled with [125I]T3 sedimented as a 4 S peak on a glycerol gradient. No receptor could be detected in the cytoplasm indicating its proper translocation to the nuclear compartment. An oligonucleotide containing a palindromic thyroid hormone response element is specifically recognized and retarded in a gel-mobility-shift assay in the presence of nuclear extract of Sf9 cells expressing hThR β. These data suggest that hThR β expressed in Sf9 cells is functional and displays characteristics virtually indistinguishable from those of the thyroid hormone receptor (ThR) extracted from mammalian cells. Furthermore, the data indicate that the baculovirus expression system is adequate for large-scale production of receptor for detailed structural and functional studies.

Original languageEnglish (US)
Pages (from-to)667-675
Number of pages9
JournalJournal of Steroid Biochemistry and Molecular Biology
Volume38
Issue number6
DOIs
StatePublished - Jun 1991

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Endocrinology
  • Clinical Biochemistry
  • Cell Biology

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