使用杆状病毒过表达的人甲状腺激素受体β1的功能特性

Functional properties of human thyroid hormone receptor beta 1 overexpressed using baculovirus.

作者信息

Collingwood T N, Sydenham M, Page M J, Chatterjee V K

机构信息

Department of Medicine, University of Cambridge, Addenbrooke's Hospital, UK.

出版信息

FEBS Lett. 1991 Oct 21;291(2):315-8. doi: 10.1016/0014-5793(91)81310-5.

DOI:10.1016/0014-5793(91)81310-5

PMID:1936281

Abstract

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.

摘要

我们利用重组杆状病毒在昆虫细胞中过表达人β1甲状腺激素受体，使其达到总细胞蛋白的5%-10%。通过SDS-PAGE和蛋白质免疫印迹分析，重组蛋白迁移为一条50 kDa的条带。表达的受体与L-T3结合，解离常数（Kd）为1.3±0.4×10⁻¹⁰ M，与甲状腺激素类似物结合，亲和力顺序为：三碘乙酸（TRIAC）＞L-T3＞L-T4＞反式三碘甲状腺原氨酸（rT3）。凝胶阻滞试验显示受体与甲状腺激素反应元件（TRE）有高度特异性结合，配体的存在会对其产生影响，抗生物素蛋白-生物素复合物DNA分析表明这种相互作用的Kd为6.2±2.0×10⁻¹⁰ M。这些结果表明hTRβ具有高水平表达，且具有真实的激素和DNA结合特性。