用酵母双杂交技术研究IGF-1与其结合蛋白3的相互作用

The Interaction of IGF-1 and IGF Binding Protein 3 in the Yeast Two-hybrid System

为了探讨胰岛素样生长因子结合蛋白 3(IGFBP- 3)分子上的胰岛素样生长因子 1 (IGF- 1 )结合位点并找出其关键氨基酸残基组成 ,首先建立了研究 IGF- 1与 IGFBP- 3相互作用的酵母双杂交模型 ,可以定性和定量地分析两个蛋白质之间的相互作用大小 ;同时利用基因体外定点突变的方法 ,对推断出的 IGF- 1结合位点中的氨基酸突变 ,经 DNA序列分析 ,构建了 5种 IGFBP- 3突变体 .然后在酵母双杂交模型中通过对报告基因活性的定量分析 ,检测 IGF- 1与各种 IGFBP- 3突变体之间相互作用的大小 .结果表明 ,IGFBP- 3分子上的 Lys2 2 2 ,Gln2 2 3突变后 ,与 IGF- 1的结合力大大下降 ,而 Arg2 2 5,Pro2 2 6,Ser2 2 7突变后也导致与 IGF- 1结合力的部分下降 .从而初步确定了IGFBP- 3分子中第 2 2 2～ 2 2 7位的氨基酸区域为 IGF- 1的结合位点 ,并且 IGFBP- 3分子上 Lys2 2 2 ,Gln2 2 3在与 IGF- 1结合中起着重要作用 .

In order to define insulin like growth factor 1(IGF 1)binding sites within IGF binding protein 3(IGFBP 3)and pinpoint the significant functional residues involved in IGF 1 binding,the interactions of IGF 1 and IGFBP 3 were verified in the yeast two hybrid system which facilitates both the qualitative detection of the interaction of proteins and quantitative analysis required for structure function studies by in vitro mutagenesis.The putative binding sites of IGF 1 within IGFBP 3 were mutated by site directed in vitro mutagenesis.Five IGFBP 3 mutants were generated after DNA sequence analysis,then the interactions of IGF 1 and variant mutant IGFBP 3 were quantified to identify the gain of function mutation in the yeast two hybrid system by assaying the β galactosidase activity.Among the several IGFBP 3 mutants,the substitutions Lys222 and Gln223 appeared to decrease dramatically IGFBP 3 association with IGF 1 compared with native IGFBP 3;the other substitutions Arg225,Pro226 and Ser227 resulted in weaker IGFBP 3 association.The result confirmed that 222 KQCRPS 227 within IGFBP 3 was the binding epitope for IGF 1 and suggested that the residues Lys222 and Gln223 on IGFBP 3 played a crucial role in binding to IGF 1.