摘要
目的 :建立简便、有效的原核表达促凋亡相关蛋白PDCD5的纯化路线 ,获得高纯度的PDCD5蛋白 ,并观察其稳定性。方法 :以包涵体形式表达的PDCD5融合蛋白 ,经包涵体洗涤、变性、复性、酶切、DEAESepharoseFastFlow和SephacrylS 2 0 0两步层析分离获得PDCD5蛋白。选用毛细管电泳方法检定蛋白质纯度 ,SDS -PAGE检定蛋白质的稳定性。结果 :经毛细管电泳方法检测PDCD5蛋白纯度为 10 0 % ,相对分子质量 15 80 0 ,等电点 5 .9。生物学活性分析PDCD5蛋白能够促进撤除细胞因子的HL 6 0细胞的凋亡 ,呈现一定的量效关系。稳定性研究发现储藏中的PDCD5蛋白不稳定 ,对温度敏感 ,4℃和 2 5℃极易降解 ,而 - 2 0℃和冻干保存则相对稳定。结论 :本研究建立的蛋白纯化方法可以获得大量、高纯度PDCD5蛋白。稳定性实验提示PDCD5宜在 - 2 0℃以下或冻干保存。
Objective:To set up an effective and simple purification method to obtain highly purified prokaryotic protein of PDCD5 and study its stability. Methods:Recombinant PDCD5 protein expressed in E. coli was accumulated as an inclusion body. After washing, the inclusion body was denatured, renatured, digested with thrombine and then purified by two steps of chromatography. The purity of the products was analyzed by capillary electrophoresis and the stability was identified by SDS-PAGE. Results:Capillary electrophoresis showed that the purity of protein was 100%, and molecular weight was 15 800 with pI 5.9. Further bioactivity assay indicated that the purified PDCD5 could enhance the apoptosis of HL 60 cells withdrawing cytokine, which was in a dose dependent manner. Stability analysis showed that the PDCD5 protein was sensitive to temperature and easy to degrade at 4 ℃ and 25 ℃. However, it was relatively stable at -20 ℃ or lyophilized. Conclusion:Highly purified and stable recombinant PDCD5 protein was obtained, which lays a foundation for the functional study and application investigation of PDCD5 .
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2003年第4期360-363,共4页
Journal of Peking University:Health Sciences
基金
国家自然科学基金 ( 3 0 2 0 0 15 7)资助~~
