核受体PPARγ结合小肽的筛选及其功能  

Bio-panning of Peptides Binding to Peroxisome Proliferator-Activated Receptor Gamma(PPARγ) and Their Bioactivity

作　　者：谢谆怡[1] 江渝[1] 叶治家[1] 彭家和[1] 何凤田[1] 曹廷兵[1] 

机构地区：[1]第三军医大学生物化学与分子生物学教研室,重庆400038

出　　处：《中国生物化学与分子生物学报》2005年第5期661-664,共4页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助项目(No.30070358);重庆市科委重点攻关项目(20016409)~~

摘　　要：为筛选与核受体过氧化物酶体增殖物激活受体γ(PPARγ)结合的功能短肽,在大肠杆菌BL21(DE3)中表达PPARγ配体结合域(LBD)的融合蛋白,并利用Ni2+-NTA离子交换树脂对表达蛋白进行纯化.以此纯化蛋白为靶,采用固体包被法对噬菌体展示随机十二肽库及环七肽库进行亲和筛选.经ELISA法鉴定特异结合的高亲和力阳性噬菌体单克隆并测序.同时利用PPARγ的配体rosiglitazone与噬菌体小肽进行竞争性结合抑制实验.最终获得与PPARγ-LBD高亲和力的十二肽3个,环七肽5个,分别含LXXLL和DXXRW(其中X为非特异氨基酸残基)保守序列.Rosiglitazone不影响噬菌体小肽与靶蛋白的结合,说明获得与配体rosiglitazone结合位点不同的目的肽.The nuclear receptors linked to either natural or synthetic ligands are currently the subject of intense drug discovery pursuit. To isolate peptides that bind to PPARγ （peroxisome proliferator-activated receptor γ）,the fusion protein PPARγ-ligand binding domain（LBD） was induced to express in E. coli BI21 （DE3）, then isolated and purified by Ni^2＋ -NTA ion exchange resin. The purified protein coated on polystyrene plate was used as the target to carry out four rounds of bio-panning. The affinity of selected recombinant phage clones was tested by ELISA,and the DNA sequences of positive clones were determined, and finally amino acid sequences of 12-peptides and C7C-peptides were deduced. Rosiglitazone,as a high-affinity PPARγ ligand, was used to carry out a competing ligand binding assay. Three positive phage clones were isolated from a 12-peptide library and five from a C7C-peptide library,and an ‘ LXXLL＇ motif was found in a 12-peptide with high affinity to PPAR7 while a consensus ＇ DXXRW＇ motif was included in C7C-peptide sequences. Rosiglitazone did not show any effect on selected peptides＇ binding to PPARγ-LBD.The peptides which could interact specifically with nuclear receptor PPARγ were obtained, and they have different binding sites on PPARγ-LBD to rosiglitazone.

关 键 词：过氧化物酶体增殖物激活受体Γ 噬菌体表面呈现 亲和筛选 

分 类 号：Q74[生物学—分子生物学] Q78