摘要
目的 :了解凋亡相关新基因TFAR19编码蛋白分子中潜在的Ser10 0 磷酸化位点与其促凋亡效应的关系。方法 :利用重叠延伸PCR定点突变技术构建TFAR19Ser10 0 →Ala10 0 突变体 ,用DNA测序技术验证 ,大肠杆菌表达 ,离子交换技术纯化重组突变蛋白 ,将其加入培养的白血病细胞株HL 6 0 ,通过PI染色 ,流式细胞仪分析 ,观察突变重组蛋白的促凋亡效应。结果 :构建成TFAR19Ala10 0 突变体 ,并经DNA测序所证实 ,在大肠杆菌中获得了高水平表达 ,Westernblot表明突变型重组蛋白与野生型具有相同的免疫学活性 ,流式细胞仪分析发现突变蛋白对撤除血清的HL 6 0细胞具有与野生蛋白相似的剂量依赖性促凋亡效应。结论 :TFAR19分子中潜在的Ser10 0 磷酸化位点对其促凋亡效应无影响 。
Objective: To explore the relation between the potential phosphorylation site Ser 100 in the novel apoptosis related molecule TFAR19 and the apoptosis accelerating effect of the TFAR19 protein. Methods: Site directed mutagenesis was used to create TFAR19 Ser 100 →Ala 100 mutant. The mutant was expressed in E.coli and the recombinant protein purified by ion exchange chromatography. Cell apoptosis was analyzed by PI labeled FACS assay. Results: High level expression of TFAR19 Ala 100 mutant protein was achieved in E.coli . The mutant TFAR19 was demonstrated by western blot using mAb against TFAR19. The purified mutant TFAR19's apoptosis accelerating effect on HL 60 cells induced by serum deprivation was dose dependent, comparable to the wild type TFAR19. Conclusion: The potential phosphorylation site Ser 100 in the TFAR19 protein does not play an important role in its apoptosis accelerating effect, implying that TFAR19 does not rely on cAMP and cGMP dependent protein kinase's activation for its biological function.
出处
《北京医科大学学报》
CSCD
2000年第4期306-309,共4页
Journal of Peking University(Health Sciences)
基金
国家自然科学基金!(39870427)资助项目
关键词
TFAR19
点突变
凋亡
氧化磷酸化
重组蛋白质
TFAR19
Point mutation
Apoptosis
Oxidative phosphorylation
Recombinant proteins/metab
