用酵母双杂交系统研究胰岛素样生长因子-1突变体与其结合蛋白-3的相互作用

Characterization of an interaction between IGF1/IGF1 mutants and IGFBP3 by using the yeast two-hybrid system

为研究胰岛素样生长因子 1(IGF1)及其突变体与IGF结合蛋白 3(IGFBP3)的相互作用 ,针对IGF1的第 3、4、15、16位氨基酸残基 ,采用定点突变的方法构建了 [Y15L16 ]IGF1和 [Q3A4Y15L16 ]IGF1。然后分别将IGF1/IGF1突变体和IGFBP3cDNA克隆至酵母表达载体pGBT9和pACT2中 ,利用酵母双杂交技术检测IGF1/IGF1突变体和IGFBP3之间的相互作用。结果表明用酵母双杂交系统检测IGF1与其结合蛋白的结合力是可行的 ,构建的这两个IGF1突变体与IGFBP3的结合力 ,与天然IGF1相比 。

In order to study the interaction between IGF1/IGF1 mutants and IGFBP3. IGF1 mutants of IGF1 and [Q3 A4 Y15 L16]IGF1 were obtained with altered sites in vitro mutagenesis system. IGF1/IGF1 mutants and IGFBP3 cDNAs were inserted into the yeast expression vectors pGBT9 and pACT2 alternatively. We used the two-hybrid assay of protein-protein interaction in the yeast Y153 to study the interaction between IGF1/IGF1 mutants and IGFBP3. The results demonstrated that the two-hybrid assay provide a facile means by which to pursue a detailed understanding of the interaction between IGF1/IGF1 mutants and IGFBP3. We also showed that IGF1 mutants had low binding affinity with IGFBP3 compared with natives.