Park J B, Ashizawa K, Parkison C, Cheng S Y
Laboratory of Molecular Biology, DCBDC, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
J Biochem Biophys Methods. 1993 Sep;27(2):95-103. doi: 10.1016/0165-022x(93)90053-q.
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.
开发了一种高效且通用的方法来纯化大量活性人β1甲状腺激素受体（h-TRβ1）。利用T7表达系统，h-TRβ1在大肠杆菌中过表达。大约80%的表达受体蛋白集中在不溶性包涵体中，约20%为可溶性形式（h-TRβ1-S）。h-TRβ1-S通过一步免疫亲和层析方便地纯化。从1升细胞培养物中可获得约0.1毫克纯化的h-TRβ1-S。纯化的h-TRβ1-S与3,3',5-三碘-L-甲状腺原氨酸结合，解离常数Ka = 2×10⁹ M⁻¹，并表现出类似物特异性。纯化的h-TRβ1-S还以不同活性与半位点中不同方向的T3反应元件（TRE）结合。此外，类视黄醇X受体增强了h-TRβ1-S与TREs的结合。这些结果表明纯化的h-TRβ1-S保留了其激素和DNA结合活性。纯化的h-TRβ1-S适用于结构和功能研究。该方法可用于纯化在昆虫细胞或酵母中表达的h-TRβ1或大鼠TRβ1。
