Analysis of proteins which specifically binds to thyroid hormone receptor β

在对甲状腺激素的抵抗中，有两种类型的甲状腺激素受体（TR）未发现任何突变的情况。我们假设存在甲状腺激素受体b的特异性辅激活因子。这种辅激活物对甲状腺激素的抵抗可能有任何异常。我们试图鉴定特定蛋白的cDNA。利用酵母双杂交系统筛选人肝脏cDNA文库。我们发现了两个cDNA克隆，No.60和93，作为tr特异性共激活子的候选。克隆60是由3种mRNA合成的片段。克隆了这些mrna的全长cdna。推导出的氨基酸序列表明蛋白质具有核转运信号。克隆60个蛋白的氨基酸序列与另一个核蛋白具有较高的同源性。为了确定克隆60蛋白是否与TRb结合，进行了下拉试验。TRb和克隆60蛋白分别表达为gst融合蛋白和His-tag融合蛋白。将两种融合蛋白与特异性抗体混合沉淀。Western blotting显示两种融合蛋白在体外结合。克隆93在体外不能表达为His-tag融合蛋白。我们正在进一步推进克隆60蛋白的分析。

There is a case where any mutations are not found in two types of thyroid hormone receptor(TR) in the resistance to thyroid hormone. We hypothesized that a specific coactivator for thyroid hormone receptor b existed. This coactivator may have any abnormality in the resistance to thyroid hormone. We tried to identify the cDNA for the specific protein. Yeast two-hybrid system was applied to screen a human liver cDNA library. We found two cDNA clones, No.60 and 93, as a candidate of TR-specific coactivator.1. A clone 60 was a fragment derived from 3 kinds of mRNA. The full-length cDNAs for these mRNAs were cloned. The deduced amino acid sequences showed that proteins had nuclear transport signals.2. In amino acid sequences of clone 60 proteins, there was a high homology sequence for another nuclear protein.3. To determine whether a clone 60 protein binds to TRb, pull-down assay was occurred. TRb and a clone 60 protein were expressed as a GST-fusion protein and His-tag fusion protein, respectively. Both fusion protein were mixed and precipitated with specific antibodies. Western blotting showed both fusion proteins bound in vitro.4. A clone 93 could not be expressed as His-tag fusion protein in vitro. We are advancing the analysis of the clone 60 proten further continuously.