利用随机多肽文库研究ZO-1中PDZ3结构域的配体结合特点

建立一种研究PDZ结构域配体结合特点的简单方法 .利用酵母双杂交技术从随机多肽文库中寻找所有可能与ZO 1中PDZ3结构域结合的C末端序列 ,从现有蛋白质数据库中检索所有具有该C末端蛋白 .利用液体培养物 β 半乳糖苷酶检测实验 ,比较文库中筛选的C末端序列和已知的PDZ3结构域结合配体———JAM的C末端 (SFLV)与PDZ3结构域结合的强弱 .共筛选到 3个阳性克隆 ,其C末端序列分别为 LGWV、 LVWV和 DEWV .前 2者属于第二类PDZ结构域 ,后者属于第三类 .蛋白质数据库检索结果表明 ,有多个蛋白质具有 LGWV、 LVWV末端 ,没有检索到任何具有 DEWV末端的蛋白质 .结合强度实验结果表明 ,它们与PDZ3结构域结合强度依次为 DEWV > LGWV > LVWV > SFLV ,说明筛选的 3个C末端除了反映ZO 1中PDZ3结构域可能的潜在结合配体外 ,也有可能成为JAM蛋白阻断性试剂甚至药物的重要组成部分之一 .利用随机多肽文库 ,可以尽可能寻找所有可能与PDZ结构域结合的C末端序列 ,大大提高了基因文库筛选的效率

Characterization of ZO-1 PDZ3 Binding Ligands with Random Peptide Library

A simple method for characteristics of PDZ domain ligands was established. C terminal peptides which interact with PDZ3 domain of ZO 1 from random peptide library were screened using yeast two hybrid technique, and then identified proteins from protein databanks swissprot and nr that contain these C termini. By liquid culture β galactosidase assay with ONPG ( O nitrophenyl β D galactopyranoside) as substrate, the interaction intensities of the C termini from the library with the PDZ3 domain were compared to that of JAM with the PDZ3 domain. Three positive clones from random peptide library were found. Sequencing results indicated that those C termini were LGWV, LVWV, and DEWV, respectively. According to the classification of PDZ domains, ZO 1 PDZ3 binding peptides LVWV and LGWV belong to class Ⅱ, and DEWV to class Ⅲ. LGWV and LVWV were found to be C termini of proteins in the database. ONPG test reveals that the interaction intensities of the three termini and JAM C terminal with the PDZ3 domain from strong to weak are DEWV> LGWV> LVWV> SFLV, which indicated that the three C termini could be used as research reagents or even drugs to block the function of JAM. Using random peptide library to find all the potential PDZ binding C termini can reduce the workload of screening many cDNA libraries, and increase the efficiency.