应用酵母双杂交技术筛选HERG钾通道相互作用蛋白

Identification of Cardiac HERG Channel Interacting Proteins Using the Yeast Two-Hybrid Technique

目的筛选心肌细胞内哪些蛋白质与HERG钾通道存在相互作用。方法 (1)通过PCR方法扩增编码心脏herg基因N端的cDNA片段(HERG-NT,aa 1-403)和C端的cDNA片段(HERG-CT,aa 669-1159),分别克隆进入pGBKT7载体,构建pGBKT7-herg-NT和pGBKT7-herg-CT诱饵质粒;(2)将被诱饵质粒转化的AH109分别涂布于SD/-Trp、SD/-Trp/-His、SD/-Trp/-Ade和SD/-Trp/X-α-Gal平板上,以观察诱饵蛋白是否自我激活报告基因;(3)将已被诱饵质粒转化的AH109分别与预转染于Y187的人类心脏cDNA文库进行酵母双杂交,分离阳性克隆中的cDNA,并测序。结果 (1)成功构建诱饵载体pGBKT7-herg-NT和pGBKT7-herg-CT,测序结果表明herg-NT和herg-CT都和"GAL4 DNA-BD-C-Myc"在同一读框,没有PCR引起的突变;(2)"诱饵"蛋白HERG-NT和HERG-CT均未能自我激化报告基因Ade2、Mel1和LacZ,弱激活报告基因His3,应用5mmol/L的3-AT可以有效抑制HERG-NT或HERG-CT所引起的组氨酸表达;(4)经过严格筛选后,发现Caveolin-1、FHL2和PTPN12等15种蛋白与HERG-NT存在相互作用,Myotrophin等8种蛋白与HERG-CT相互作用。结论成功应用酵母双杂交技术,以HERG钾通道蛋白的氨基端或羧基端为诱饵蛋白,筛选HERG钾通道的相互作用蛋白(Caveolin-1、FHL2、PTPN12、Myotrophin等),这些蛋白质可能与HERG存在相互作用。

Objective To screen the cardiac proteins which interact with HERG channel. Methods： （1） The N-teminal fragment （herg-NT, aa 1-403） and the C-teminal fragment （herg-CT, aa 669-1159） of herg gene were PCR amplified, and then were cloned into the pGBKT7, respec- tively, to construct the bait plasmids （pGBKT7-herg-NT and pGBKT7-herg-CT）. （2） The AH109 transformed with pGBKT7-herg-NT or pGBKT7- herg-CT was plated on the different synthetic dropout （SD） plates, SD/-Trp, SD/-Trp/-His, SD/-Trp/-Ade and SD/-Trp/X- ct -GAL, to determine whether the baits themselves auto-activated the reporter genes; （3） The AH109 transformed with the pGBKT7-herg-NT or pGBKT7-herg-CT mated with Y187 pre-transformed with human heart cDNA library, and plasmids extracted from positive clones were sequenced. Results： （1） The bait plas- raids （pGBKT7-herg-NT and pGBKT7-herg-CT） were constructed; either the herg-NT or the herg-CT was in the same frame with the Gal4 DNA binding domain. （2） There was no auto-activation by the bait protein, but weak leaky His3 expression was found, which was inhibited by 5 mmol/L 3-AT. （3） 15 proteins, including Caveolin-1, FHL2, PTPN12, etc., were identified as the potential HERG-NT partner, 8 proteins, including myotrophin, etc., were identified as the potential HERG-CT partner. Conclusion： The yeast two hybrid can be use to screen the HERG interacting proteins using the different HERG domain as bait, respectively. Caveolin-1, FHL2, PTPN 12 and Myotrophin may be the HERG interacting protein.