Purification and properties of thyroid hormone receptor beta 1 expressed in Escherichia coli as a fusion protein.
Thyroid hormone receptor binds to specific DNA sequences and acts as a hormone-dependent transcriptional regulator. The protein can form homodimers, or heterodimers with the related 9-cis-retinoic acid receptor (RXR) or retinoic acid receptor (RAR) receptor families, leading to complex patterns of regulation. To obtain relatively large quantities of the receptor for biochemical studies, we have inserted the cDNA for human thyroid receptor beta into a variant of the pGEX vector (pGEX-KG) and produced the protein in Escherichia coli as a fusion with glutathione-S-transferase. Conditions for protein production, isolation on glutathione agarose, and thrombin cleavage to generate active receptor were developed. Final yields were approximately 1 mg/liter of culture. Scatchard plots of 125I-triiodothyronine binding data revealed a single class of sites with a Kd of 0.1 nM. An overlay assay was established to measure protein-protein binding and used to show a direct interaction with bacterially expressed RXR receptor. Binding of the purified receptor to DNA response elements measured in a DNA binding assay was increased by RXR to different extents, depending on the DNA sequence. This preparation will be useful in exploring the mechanisms of receptor activity.
Ball
E H
EH
Department of Biochemistry, University of Western Ontario, London, Canada.
Shephard
L B
LB
Gill
G N
GN
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D011988:Receptors, Thyroid Hormone / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism
D004268:DNA-Binding Proteins; D018168:Receptors, Retinoic Acid; D011988:Receptors, Thyroid Hormone; D011993:Recombinant Fusion Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014284:Triiodothyronine; D004247:DNA; D005982:Glutathione Transferase