Expression of the rat alpha 1 thyroid hormone receptor ligand binding domain in Escherichia coli and the use of a ligand-induced conformation change as a method for its purification to homogeneity.
The rat alpha 1 thyroid hormone receptor (rTR alpha 1) mediates hormone-dependent gene regulation by utilizing several distinct structural domains, including those containing DNA and ligand binding sites. Binding of the hormone to the ligand binding domain (TR-LBD) induces conformational changes in the receptor that are involved in affecting the receptor's transcriptional regulatory and other functions. A 33-kDa protein fragment (Met122-Val410) of rTR alpha 1, which includes the entire TR-LBD, was expressed in Escherichia coli, yielding typically 1.5 mg of soluble TR-LBD/liter of bacteria. The protein was purified to > 99% homogeneity with a final yield of 24% by hydrophobic interaction, DEAE anionic exchange, and heparin cationic exchange chromatographic steps. The Kd of the purified TR-LBD for 3,3',5-triiodo-L-thyronine (T3) was 0.06 nM, identical to that for full-length rTR alpha 1. T3 analogs had affinities consistent with values obtained for full-length rTR alpha 1. In all three chromatography steps, TR-LBD prebound to [125I]T3 eluted earlier than the unliganded TR-LBD, like the full-length receptor. These studies indicate that the binding affinity and specificity of the TR-LBD are similar to those of the intact rTR alpha 1 and that the ligand-induced conformational changes occur in the LBD itself. These studies also provide methodology for obtaining milligram quantities of protein useful for biochemical and biophysical studies of the thyroid hormone receptor and its ligand-induced changes.
Apriletti
J W
JW
Metabolic Research Unit, University of California, San Francisco 94143-0540, USA.
Baxter
J D
JD
Lau
K H
KH
West
B L
BL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002845:Chromatography / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D008024:Ligands; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D011988:Receptors, Thyroid Hormone / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013963:Thyroid Hormones / Q000378:metabolism
D008024:Ligands; D010446:Peptide Fragments; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins; D013963:Thyroid Hormones