Pichia酵母表达重组人可溶性TRAIL的纯化与生物活性Purification and Bioactivity of Recombinant Human Soluble TRAIL Expressed in Pichia pastoris
李茹冰,傅泳航,李继东,佟明华,杨联萍,孔祥平
LI Ru-bing,FU Yong-hang,LI Ji-dong,et al (The 458th Hospital of PLA,Guangzhou 510600,China)
摘要(Abstract)：
目的分离纯化Pichia酵母表达的重组人可溶性TRAIL,并检测其生物活性。方法以Pichia酵母分泌型表达TRAIL,采用硫酸铵沉淀和柱层析纯化可溶性TRAIL蛋白。以SDSPAGE、ESIMS和Westernblot法鉴定其纯度及特异性。以细胞透射电镜、DNALadder和MTT法检测其诱导肿瘤细胞凋亡活性。结果目的蛋白以可溶形式存在,蛋白相对分子质量22000,纯化后纯度95%。Westernblot证实为TRAIL蛋白。电镜、DNALadder和MTT均证实其具有诱导肿瘤细胞凋亡活性。结论Pichia酵母表达rhsTRAIL可用柱层析法纯化,并具有诱导肿瘤细胞凋亡的生物活性。
Objective To separate and purify the recombinant human soluble TRAIL(rhsTRAIL) expressed in Pichia pastoris and study its bioactivity.Methods Express rhsTRAIL in Pichia pastoris secretary expression system and purify by ammonium sulfate precipitation and column chromatography.Identify the purity and specificity of purified rhsTRAIL by SDS-PAGE,electrospray ionization-mass specectrometry(ESI-MS) and Western blot.Determine the bioactivity of rhsTRAIL in inducing the apoptosis of tumor cells by transmission electron microscopy,DNA ladder and MTT method.Results The expressed product with a relative molecular weight of 22 000 existed in a soluble form,reached a purity of 95% after purification and was identified as TRAIL protein by Western blot.Transmission electron microscopy,DNA ladder and MTT method showed bioactivity of rhsTRAIL in inducing the apoptosis of tumor cells.Conclusion The rhsTRAIL expressed in Pichia pastoris could be purified by column chromatography and showed bioactivity in inducing the apoptosis of tumor cells.
关键词(KeyWords)：
肿瘤坏死因子相关凋亡诱导配体;Pichia酵母;生物活性
TNF-related apoptosis inducing ligand(TRAIL);Pichia pastoris;Bioactivity
基金项目(Foundation):
作者(Author):
李茹冰,傅泳航,李继东,佟明华,杨联萍,孔祥平
LI Ru-bing,FU Yong-hang,LI Ji-dong,et al (The 458th Hospital of PLA,Guangzhou 510600,China)
DOI: 10.13200/j.cjb.2006.03.98.lirb.031
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