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目的 通过分析端粒主要结合蛋白端粒重复因子2 (TRF2 )与p53的体外结合,探讨p53通过端粒途径调节细胞增殖、衰老和凋亡的分子机制。方法 4种不同的p53 谷胱甘肽S转移酶(glutathioneS transferase,GST)融合蛋白和GST经大肠杆菌表达、谷胱甘肽SepharoseTM4B纯化,其中的人重组p53包括野生型(1~393)、C端缺失体p53N5(2~293)、N端缺失体p53N5 (95 ~393)、第175位氨基酸突变体(R→H)。将各纯化蛋白和人乳腺癌细胞MCF 7的细胞蛋白进行体外结合反应(pulldown),Westernblot检测反应物中p53和TRF2的结合。结果 纯化的GST和p53 GST融合蛋白纯度均在90%以上,且相对分子质量与预计的完全一致。TRF2的Westernblot显示:野生型p53和p53 R175H均能沉淀MCF 7中的TRF2,且结合力相似,而单独的GST则无沉淀TRF2的作用。与野生型p53和p53R175H相比,p53 2C与TRF2的结合力相对增加,p53N5与TRF2的结合力相对大大减弱。结论 p53和TRF2可以进行直接而特异的体外结合,且其结合部位在p53的C端( 293 ~393)。p53和TRF2的C端依赖性结合可能与端粒动态变化所诱导的细胞活动有关。
OBJECTIVE: To investigate the molecular mechanisms by which p53 regulates cell proliferation, aging and apoptosis through the telomere pathway by analyzing the in vitro binding of telomeric repeat factor 2 (TRF2) to p53 in vitro. Methods Four different glutathione S transferase (GST) fusion proteins and GST were expressed in Escherichia coli and purified with glutathione SepharoseTM 4B. Among them, human recombinant p53 included wild type (1-393), C The deletion of p53N5 (2 ~ 293), N-terminal deletion of p53N5 (95 ~ 393), the first 175 amino acid mutations (R → H). Each purified protein and the cellular protein of human breast cancer cell MCF 7 were subjected to an in vitro pulldown, and the binding of p53 and TRF2 in the reaction was detected by Western blot. Results The purity of the purified GST and p53 GST fusion proteins was above 90%, and the relative molecular mass was exactly the same as expected. Western blotting of TRF2 showed that both wild-type p53 and p53 R175H precipitated TRF2 in MCF7 with similar binding, whereas GST alone did not precipitate TRF2. Compared with wild-type p53 and p53R175H, the binding affinity of p53 2C to TRF2 is relatively increased, and the binding affinity of p53N5 to TRF2 is relatively greatly weakened. Conclusion p53 and TRF2 can be directly and specifically bound in vitro, and the binding site is at the C-terminal of p53 (293-393). The C-terminal-dependent binding of p53 and TRF2 may be related to the cellular activities induced by the dynamic changes of telomeres.