【摘要】 LPTS基因是利用定位候选克隆策略克隆的一个新的肝相关候选肿瘤抑制基因。LPTS基因编码一个全长为328氨基酸的蛋白质(LPTS-L),该蛋白具有抑制细胞端粒酶活性的功能。为了进一步研究LPTS-L蛋白的结构与功能,利用DNA重组技术,将LPTS-L的cDNA克隆到表达载体pET-24a中构建重组克隆pET-24-LPTS,并在大肠杆菌BL-21中进行融合表达,获得可溶形式的LPTS-L融合蛋白。采用Ni Sepharose4B柱亲和层析,可以获得纯度较高的蛋白,但不适合大量制备。通过设计引物去掉了pET-24a载体上的6×His tag将LPTS-L基因进行了非融合表达,然后采用磷酸纤维素P11阳离子交换层析纯化LPTS-L蛋白,纯度可达到55%。再经Sephadex G-100凝胶过滤,LPTS-L蛋白的纯度可达到80%。Western blot实验显示经纯化后的LPTS-L蛋白可与兔抗GST-LPTS-L的多抗发生特异性结合。采用TRAP法测定蛋白质活性,结果显示纯化得到的LPTS-L蛋白可抑制端粒酶的活性,与采用Ni Sepharose4B纯化获得的LPTS-L融合蛋白比较,其抑制效率基本一致。因此,所建立的技术可以有效地制备LPTS-L蛋白。更多还原

【Abstract】 The gene for LPTS is originally cloned as a human liver-related putative tumor suppressor(LPTS)gene that encodes a full length protein of 328 amino acids(LPTS-L).LPTS-L is also identified as a telomerase inhibitor to regulate telomere length in the cells.To facilitate the functional and structural studies of LPTS-L protein,the cDNA for LPTS-L was cloned into the expression vector pET-24 in frame to generate a recombinant plasmid pET-24-LPTS.The LPTS-L protein was expressed in E.coli BL21 solublely,and purified by Ni Sepharose affinity chromatography which,however,is not fit for large scale protein purification.The gene of LPTS-L was then PCR amplified to remove the 6×His tag,and cloned into pET-24a.The non-fusion protein of LPTS-L was expressed in E.coli BL21,and purified by phosphocellulose P11 chromatography.The purity of LPTS-L protein was about 55% after that procedure,and arrived at 80% after second purification by Sephadex G-100 chromatography.Western Blotting analysis showed that the band reflects the specific binding of anti-LPTS antiserum against the purified LPTS-L protein.The TRAP assay was performed to detect the telomerase inhibitory activity of LPTS-L protein in vitro.It was observed that the purified LPTS-L inhibited the activity of telomerase greatly,similarly with that of LPTS-L protein purified by Ni Sepharose 4B.Our results suggest that phosphocellulose P11 plus Sephadex G-100 chromatography could substitute for Ni Sepharose 4B affinity chromatography for preparation of purified LPTS-L protein.Through this study,a technique for preparation of LPTS-L protein in a large scale is established.更多还原