Functional properties of human thyroid hormone receptor beta 1 overexpressed using baculovirus.
We have overexpressed the human beta 1 thyroid hormone receptor in insect cells using a recombinant baculovirus to a level of 5-10% of total cellular protein. The recombinant protein migrates as a 50 kDa band by SDS-PAGE and Western blot analysis. The expressed receptor binds to L-T3 with a Kd of 1.3 +/- 0.4 x 10(-10) M and to thyroid hormone analogues with an affinity hierarchy of TRIAC greater than L-T3 greater than L-T4 greater than rT3. Gel retardation assays show highly specific receptor binding to a TRE which is modified by the presence of ligand and avidin-biotin complex DNA analysis shows a Kd of 6.2 +/- 2.0 x 10(-10) M for this interaction. These results indicate high level expression of hTR beta with authentic hormone and DNA binding properties.
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, UK.
T N
TN
Collingwood
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M
Sydenham
M J
MJ
Page
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VK
Chatterjee
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics; D011988:Receptors, Thyroid Hormone / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins