利用酵母双杂交系统筛选Pak4相互作用蛋白质

Screening of Pak4 Interacting Protein by Yeast Two-hybrid System

目的利用酵母双杂交系统从人胎脑cDNA文库中筛选信号转导分子Pak4相互作用蛋白。方法将Pak4激酶域序列克隆到酵母表达载体pGBKT7构建诱饵质粒。质粒转化到酵母感受态AH109和Y187中验证蛋白表达。将人胎脑cDNA文库转化酵母Y187,与已转化诱饵质粒的AH109酵母进行交配实验。交配子在SD/-Leu/-Trp/-His和SD/-Leu/-Trp/-His/-Ade/X-α-gal平板上筛选,阳性克隆质粒酶切鉴定。结果成功构建了pGBKT7-Pak4 KD诱饵质粒,Western blot证实其在酵母菌AH109和Y187中表达,筛选了多个与Pak4相互作用的阳性转化子。结论利用酵母双杂交系统筛选出Pak4相互作用蛋白质。

Objective To screen the p21 -activated kinase 4 （ Pak4 ） interacting proteins from the human fetal brain cDNA library by using yeast two-hybrid system. Methods The kinase domain sequence of Pak4 （Pak4 KD ） fused to yeast-expressing vector pGBKT7 （pGBKTY-Pak4 KD） as ＂bait＂ was transformed into the yeast competent cells AHI09 and Y187. Protein expression of the ＂bait＂ in yeast cells was identified by Westem blot. After pGBKT7-Pak4 fusion protein was expressed in the AH109 yeast strain, a yeast two-hybrid screening was performed by mating AH109 with Y187 containing a human fetal brain cDNA library plasmid. The mating transformants were selected on SD/-Leu/-Trp/-His and SD/- Leu/-Trp/-His/-Ade/X-α-gal plates. The constructs of the positive clones were digested with Bgl Ⅱ Results The Pak4 KD fragments were successfully subcloned into pGBKTT. The results of Western blot showed that the ＂bait＂ was expressed in yeast strains Y187 and AH109. Several positive clones which interacted with Pak4 were successfully screened from huanl fetal brain cDNA library. Conclusion Several Pak4 interacting proteins could be screened by using yeast two-hybrid system.