AF-2结构域突变的SF1酵母表达质粒的构建及其与PNRC相互作用分析

Construction of yeast expression plasmids of SF1 with or without AF-2 domain and assays for its interaction with PNRC

目的 探讨激活功能 2 (AF 2 )结构域在孤儿核受体辅活化富含脯氨酸的受体辅调节蛋白 (PNRC)相互作用的意义。方法 采用PCR和DNA重组技术构建了野生型SF1和AF 2结构域突变的SF1的酵母表达质粒pGBT9 SF1/HBDD和pGBT9 SF1/HBD/AF 2del并采用酵母双杂合分析比较了它们与PNRC的相互作用强弱。结果 SF1/HBD能与野生型PNRC及其N 末端缺失突变体PNRC2 70 3 2 7,PNRC2 78 3 0 0产生较强的相互作用 ,而AF 2缺失的SF1/HBD不与这些PNRC片段发生相互作用。结论 功能性的AF 2结构域是SF1与PNRC相互作用所必须的。

Objective To investigate the role of AF 2(Activation function 2)domain in the interaction between SF1 (Steroidogenic factor 1) and nuclear receptor coactivator PNRC (Proline rich nuclear receptor coregulatory protein). Methods The yeast expression plasmids of SF1/HBD and AF 2 domain deleted SF1/HBD were constructed and tested for interaction with PNRC. Results SF1/HBD interacted strongly with wild type of PNRC and its N terminus deletion mutants(PNRC 278 300 and PNRC 270 327), but SF1/HBD with deleted AF 2 domain could not. Conclusion The results suggest that functional AF 2 domain is required for the interaction between PNRC and SF1.