应用酵母双杂交系统筛选新基因Collectrin相关蛋白

Collectrin是在小鼠 5 6肾切除后 ,在肾小球的高滤过、高增生期分离克隆的一个新基因 .通过酵母双杂交系统从人肾脏cDNA文库中筛选与collectrin相互作用的蛋白 ,可以为该基因的功能研究提供线索 .构建collectrin的真核表达载体collectrin pGBKT7 c myc ,转化酵母菌AH10 9.Western印迹证实 ,collectrin蛋白能够在酵母中正常表达 ,对酵母细胞无毒性 ,不存在自激活现象 .将AH10 9 collectrin pGBKT7 c myc与转化了成人肾脏cDNA文库的酵母菌Y187接合 ,共筛选到 5个与细胞代谢有关的蛋白 ,包括鞘磷脂激活蛋白、精氨琥珀酸合成酶、氨基酸转运蛋白XAT2、NADH脱氢酶 1和金属硫蛋白 2A .由此推论 ,collectrin可能通过与细胞内某些酶类相互作用而影响细胞代谢 ,为新基因collectrin的功能研究提供了重要线索 .

Screening for Proteins Interacting with Novel Gene Collectrin in Adult Human Kidney cDNA Library by Yeast Two Hybrid System

A novel kidney specific gene collectrin, was recently identified in 5/6 ablated kidneys at hypertrophic phase. To screen for proteins interacting with collectrin, the yeast two-hybrid system was performed. The intracellular domain of collectrin was fused to a DNA-binding domain as “Bait" and the yeast strain AH109 was transformed with collectrin-pGBKT7/c-myc. Western blot analysis revealed that collectrin protein was expressed normally and there was no self-activation and toxicity on yeast strain AH109. Then the AH109/collectrin-pGBKT7/c-myc were mated with the yeast strain Y187 pretransformed with human kidney cDNA library to screen for interacting proteins with collectrin. Eighteen proteins that interacted with collectrin from human kidney library were identified. Five have homology with known proteins, including sphingolipid activator protein, argininosuccinate synthetase, amino acid transporter XAT2, NADH dehydrogenase 1 and metallothionein 2A. These results revealed that collectrin might interact with some proteins, and interfere with cell material or energy metabolism.