从噬菌体展示随机12肽库中筛选与IRS-1PH结构域相结合的多肽模体  

Identification of the peptide motif that can bind to IRS-1's PH domain by panning of phage displayed peptide library

作　　者：韩炯[1] 张晓光[1] 刘新平[1] 药立波[1] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,西安710032

出　　处：《生物技术通讯》2004年第5期437-440,共4页Letters in Biotechnology

基　　金：国家自然科学基金项目(30000067)

摘　　要：将胰岛素受体底物-1(IRS-1)的PH结构域(pleckstrinhomologydomain)编码序列克隆到融合表达载体pRSETA中,阳性克隆经IPTG诱导,表达出氨基端带6个连续组氨酸残基的融合蛋白。经检测,表达的目的蛋白一部分以可溶形式存在。利用Ni-NTA金属螯合亲和层析法在非变性条件下对表达的目的蛋白进行纯化,纯度大于98%。将纯化的目的蛋白包被于聚苯乙烯平皿上作为靶蛋白,经过4轮淘筛,得到能够与IRS-1的PH结构域相结合的重组噬菌体克隆;从中随机挑选出50个克隆进行DNA序列测定,对获得的短肽序列进行了分析。并通过ELISA检测了这些克隆与IRS-1的PH结构域的结合活性。The pleckstrin homology (PH) domain plays an important role in the network of cellular signal transduction. To identify the peptide motif that can bind to PH domain, the gene encoding the PH domain of insulin receptor substrate 1(IRS-1) was cloned into the fusion expression plasmid vector pRSET A. Then the recombinant plasmids were transformed into competent cell of E.coli JM109(DE3). After induction with IPTG, the positive strain expressed the fusion protein with a hexahistidyl tag on the N-terminal under the control of T7 promoter. SDS-PAGE analysis showed that some of the expressed protein existed in a soluble form. The protein was purified under natural conditions by Ni-NTA metal chelate chromatography. The purity was up to 98%. The purified-protein-coated polystyrene plate was used to screen recombinant phages that could bind onto it. After four rounds of affinity screening, 50 phages that could bind specifically with the PH domain of IRS-1 were selected from a random phage-displayed twelve-peptide library. The peptide sequences of the positive phage clones were analyzed.

关 键 词：IRS-1 表达 阳性克隆 PH 靶蛋白 肽库 短肽 结构域 模体 IPTG 

分 类 号：Q78[生物学—分子生物学] R587.2[医药卫生—内分泌]