One-step affinity purification protocol for human telomerase
One-step affinity purification protocol for human telomerase
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DOI：
10.1093/nar/26.13.3311
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发表时间：
1998-07-01
影响因子：
14.9
通讯作者：
Damm, K
中科院分区：
文献类型：
--
作者：
Schnapp, G;Rodi, HP;Damm, K
人端粒酶是一种核糖核蛋白(RNP)酶，由蛋白质组分和通过添加TTAGGG重复序列催化端粒延长的RNA模板组成。端粒酶功能与衰老和癌细胞永生化有关。我们报道了一种快速高效的一步纯化方法，可以从人细胞中获得高活性的端粒酶。纯化是基于核抽提物的亲和层析，其中反义寡核苷酸互补于人端粒酶RNA组分的模板区域。结合的端粒酶在温和的条件下用置换寡核苷酸洗脱。：得到的亲和纯化的端粒酶在PCR扩增的端粒酶分析中是活跃的。纯化的端粒酶复合体的分子质量类似于550 kDa，而在未分离的核提取液中端粒酶RNP的分子质量类似于1000 kDa。该纯化方法为研究人端粒酶的功能和结构提供了一种快速有效的工具。
Human telomerase is a ribonucleoprotein (RNP) enzyme, comprising protein components and an RNA template that catalyses telomere elongation through the addition of TTAGGG repeats. Telomerase function has been implicated in aging and cancer cell immortalization. We report a rapid and efficient one-step purification protocol to obtain highly active telomerase from human cells. The purification is based on affinity chromatography of nuclear extracts with antisense oligonucleotides complementary to the template region of the human telomerase RNA component. Bound telomerase is eluted with a displacement oligonucleotide under mild conditions. The:resulting affinity-purified telomerase is active in PCR-amplified telomerase assays. The purified telomerase complex has a molecular mass of similar to 550 kDa compared to the similar to 1000 kDa determined for the telomerase RNP in unfractionated nuclear extracts. The purification protocol provides a rapid and efficient tool for functional and structural studies of human telomerase.