Soluble domains of telomerase reverse transcriptase identified by high-throughput screening
Soluble domains of telomerase reverse transcriptase identified by high-throughput screening
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DOI：
10.1110/ps.051532105
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发表时间：
2005-08-01
期刊：
影响因子：
8
通讯作者：
Cech, TR
中科院分区：
文献类型：
--
作者：
Jacobs, SA;Podell, ER;Cech, TR
端粒酶是一种核糖核蛋白复合物，负责延长真核生物染色体的末端。由于无法产生大量的重组蛋白，这种酶的结构和生物物理研究受到了限制。在这里，我们使用gfp融合系统进行高通量筛选，以绘制在大肠杆菌中以可溶性形式过表达的嗜热四膜虫TERT（端粒酶逆转录酶）蛋白区域。鉴定的许多可溶性蛋白结构域与从多个序列比对中推断的结构域不一致，因此筛选荧光菌落提供了不易获得的信息。该方法揭示了一个重要的、独立折叠的n端结构域，该结构域以高产量表达和纯化，并被发现适合于结构分析。这些结果为今后TERT的结构和生物物理研究提供了工具。
Telomerase is a ribonucleoprotein complex responsible for extending the ends of eukaryotic chromosomes. Structural and biophysical studies of this enzyme have been limited by the inability to produce large amounts of recombinant protein. Here we perform a high-throughput screen to map regions of the Tetrahymena thermophila TERT (Telomerase Reverse Transcriptase) protein that are overexpressed in a soluble form in Escherichia coli using a GFP-fusion system. Many of the soluble protein domains identified do not coincide with domains inferred from multiple sequence alignment, so screening for fluorescent colonies provided information not otherwise readily obtained. The method revealed an essential, independently folded N-terminal domain that was expressed and purified with high yield and found to be suitable for structural analysis. These results provide a tool for future structural and biophysical studies of TERT.