酵母双杂交筛选PMEPA1互作蛋白

Screening for PMEPA1 Interacting Protein Using Yeast Two-hybrid System

采用酵母双杂交技术筛选与前列腺跨膜蛋白PMEPA1相互作用的蛋白质,构建了pGBKT7-PMEPA1诱饵质粒,获得与PMEPA1互作蛋白,GST pull-down实验确定2种蛋白间的相互作用.构建了诱饵载体转化酵母菌Y2HGold,对酵母宿主无毒性、无自激活活性.得到阳性克隆Dynactin6,GST pull-down实验证实PMEPA1与Dynactin6蛋白间相互作用.推测蛋白Dynactin6可能参与调控PMEPA1在细胞的运输.

To screen the candidate protein interacting with prostate transmembrane protein, androgen induced 1 （PMEPA1）. The bait plasmid pGBKT7 -PMEPA1 was constructed and transformed into yeast strain Y2H Gold, and then validated by auto -activation and toxicity assays. A universal human library was used for screening with the bait plasmid pGBKT7 - PMEPA1, and positive clones were selected after screening and co - transformation identification. The interaction between two proteins was further analyzed by GST pull - down. The bait plasmid pG- BKT7 -PMEPA1 was successfully constructed. No auto -activation and toxicity were detected. A target protein of Dynactin6 was fished out and GST pull -down assay confirmed the interaction between PMEPA1 and Dynactin6. Dynactin6 was identified as a PMEPA1 interacting protein using yeast two -hybrid system, suggesting that Dynac- tin6 might be involved in regulating intracellular protein transport of PMEPA1. These results will lay a foundation to further study the biological function of PMEPA1.