摘要
为分离纯化人类端粒酶复合体并对其蛋白质组分进行分析 ,自行设计一套特异的以端粒重复序列为配体 ,以亲和磁珠为介质 ,以竞争性碱基序列为洗脱原则的寡核苷酸亲和纯化法 ,对He La细胞端粒酶复合体进行分离纯化 .并采用近年发展起来的主要用于检测序列特异性 DNA结合蛋白的凝胶迁移阻滞分析法对纯化产物进行鉴定分析 .应用 SDS- PAGE对纯化蛋白质亚基组分进行分析与鉴定 ,并采用电泳迁移率和已知蛋白质分子质量标准的对数作图分析测得所得蛋白质亚基成分的相对分子质量 .结果表明 ,纯化产物以 TRAP法检测酶活性可见典型的梯形条带 ;比活性为每 mg蛋白质的 cpm值为 1 80× 1 0 9,纯化倍数 1 80 0 ,得率 90 % .凝胶迁移阻滞法分析显示特异的凝胶迁移阻滞性电泳条带 ;以 SDS- PAGE检测得到 4种蛋白质亚基成分 ,其蛋白质相对分子质量分别为 2 2 0 ku、2 1 2 ku、1 1 6ku和 43ku.由上述可见 ,采用自行设计的寡核苷酸亲和纯化法获得了人端粒酶复合体及 4种蛋白质亚基组分 .
To purify human telomerase complex and analyse its protein subunit components,an affinity purification protocol using human telomere repeat sequence as affinity ligand was designed and carried out to purify HeLa cell telomerase.A DIG gel shift protocol was performed to identify the purified component.SDS PAGE was used to analyze protein subunits of purified component.The component with telomerase activity was obtained from elution fluid.A single band on native gel and a special DIG gel shif band on X ray film were observed.Four protein subunits were observed using SDS PAGE. Their relative molecule mass are 220 ku,212 ku,116 ku and 43 ku,respectively.Human telomerase complex and its 4 protein subuints were obtained.
出处
《中国生物化学与分子生物学报》
CSCD
2000年第5期574-579,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金资助!( 3 9670 715 )
关键词
人类端粒酶
蛋白质组分
亲和层析
分离
鉴定
Human telomerase
hTR
Affinity purification
Protein subunits
DNA binding protein
