运用酵母双杂交系统筛选肌营养不良相关蛋白2(DRP2)相互作用蛋白(英文)

Identification of a dystrophin-related protein 2(DRP2) associating protein by yeast two hybridization

目的应用酵母双杂交系统筛选小鼠、大鼠、人类大脑文库中与DRP2相互作用的蛋白,分析中枢神经系统中DRP2分子复合体的组成成分。方法设计3个含有DRP2不同蛋白结构域的诱饵质粒,在完成诱饵质粒的自激活性的鉴定后,对小鼠、大鼠、人类脑MATCHMAKER cDNA文库进行筛选。结果运用DRP2蛋白N-Bait筛选大鼠脑MATCHMAKER cDNA文库,笔者获得了一个阳性文库质粒。该质粒的插入子编码一种SNAP25相互作用的蛋白质Scoilin。Scoilin又被命名为Zwint-1蛋白。同时笔者还确定了这两种相互作用蛋白质的精确结构域:DRP2蛋白以其含有两个Spectrin重复和一个WW蛋白结构域的氨基端与Scoilin(Zwint-1)全蛋白相互作用。结论应用酵母双杂交,笔者于大鼠大脑中找到了DRP2的相互作用的蛋白质Scoilin(Zwint-1),并且确定了这两种相互作用蛋白质的精确结构域。

[Objective] To reveal the candidate association members of the DRP2 molecular complex in the central nervous system, the mouse, rat and human brain MATCHMAKER cDNA library was screened by yeast two-hybridization. [Methods] Three different baits spanning various domains of the DRP2 protein were constructed. The three baits were used to screen the mouse, rat and human brain MATCHMAKER cDNA library after being fully validated. [Results] A positive interaction protein Scoilin with DRP2 was found via the rat brain library screening with the N-bait. The Scoilin is also named Zwint-1 protein. The intricate interaction domains between these two proteins were further located： via associating with the two spectrin repeats and the WW domain at the N-terminus of the DRP2 protein, the full length Scoilin （Zwint-1） protein and DRP2 are associated with each other.. [Conclusions] Scoilin （Zwint-1） protein ,an association member of the DRP2 protein, was found in the brain via Y2H, and the intricate interaction domains in these two proteins were further located.