One-step affinity purification protocol for human telomerase.

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 approximately 550 kDa compared to the approximately 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.人类端粒酶是一种核糖核蛋白（ribonucleoprotein, RNP）酶，由蛋白质组分与RNA模板构成，可通过添加TTAGGG重复序列催化端粒延长。端粒酶的功能与衰老及癌细胞永生化密切相关。本研究报道了一种快速高效的单步纯化方案，可从人类细胞中获取高活性端粒酶。该纯化方案基于核提取物的亲和层析技术，使用与人类端粒酶RNA组分模板区域互补的反义寡核苷酸作为配基。结合在层析介质上的端粒酶可在温和条件下通过置换寡核苷酸进行洗脱。经亲和纯化得到的端粒酶在PCR扩增的端粒酶活性检测实验中具有活性。经纯化的端粒酶复合物的分子量约为550 kDa，而未分级核提取物中端粒酶RNP的分子量约为1000 kDa。该纯化方案为人类端粒酶的功能与结构研究提供了一种快速高效的研究工具。