One-step immunoaffinity purification of human beta 1 thyroid hormone receptor with DNA and hormone binding activity.
An efficient and versatile method to purify large amounts of active human beta 1 thyroid hormone receptor (h-TR beta 1) was developed. Using a T7 expression system, h-TR beta 1 was overexpressed in Escherichia coli. Approx. 80% of the expressed receptor protein was concentrated in the insoluble inclusion bodies and approximately 20% was in the soluble form (h-TR beta 1-S). h-TR beta 1-S was conveniently purified by one immunoaffinity chromatographic step. From 1 l of cell culture, approx. 0.1 mg of purified h-TR beta 1-S was obtained. The purified h-TR beta 1-S binds to 3,3',5-triiodo-L-thyronine with a Ka = 2 x 10(9) M-1 and exhibits analog specificity. The purified h-TR beta 1-S also binds to T3 response elements (TRE) with different orientation in the half-sites with differential activity. In addition, binding of h-TR beta 1-S to TREs was enhanced by retinoid X receptor. These results indicate that the purified h-TR beta 1-S retains its hormone and DNA binding activity. The purified h-TR beta 1-S is suitable for structural and functional studies. This method could be used to purify h-TR beta 1 or rat TR beta 1 expressed in insect cells or yeast.
Park
J B
JB
Laboratory of Molecular Biology, DCBDC, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Ashizawa
K
K
Parkison
C
C
Cheng
S Y
SY
D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D004247:DNA / Q000378:metabolism*; D008969:Molecular Sequence Data; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D018168:Receptors, Retinoic Acid; D011988:Receptors, Thyroid Hormone / Q000302:isolation & purification* / Q000378:metabolism*; D047488:Retinoid X Receptors; D012995:Solubility; D013963:Thyroid Hormones / Q000378:metabolism*; D014157:Transcription Factors; D014284:Triiodothyronine / Q000378:metabolism
D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D011988:Receptors, Thyroid Hormone; D047488:Retinoid X Receptors; D013963:Thyroid Hormones; D014157:Transcription Factors; D014284:Triiodothyronine; D004247:DNA