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Screening and Application of Ligand-mimicking Peptides

模拟配体肽的筛选及应用

基本信息

批准号：
07558228
负责人：
AOYAMA Takashi
金额：
$ 1.34万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07558228/
关键词：
Transgenic Plant Calmodulin CaM Kinase I CaM Kinase II Glucocorticoid Transcriptional Induction System CDPK Random Peptide Library 模倣ペプチド CDPKキナーゼI cAMP・レセプター蛋白質 G蛋白質

项目摘要

0ligopeptides can mimic various 3-dimensional structures depending on their amino-acid sequences. On the basis of the idea of mimicking peptides, we tried to mimic the function of a non-peptide ligand with oligopeptides in vivo. First, we constructed a novel phage-random-peptide library by modifying the vector phage Ml3mpl8.This phage library was screened for calmodulin (CaM) - binding peptides, and two oligopeptides (termed peptides 1 and 2 ; WDTVRlSF and WPSLQAlR,respectively) were identified. The two peptides differentially inhibited CaM-dependent kinase I and II (CaM kinase I and Il). Peptide 1 inhibited CaM kinase I but not CaM kinase II,whereas peptide 2 inhibited CaM kinase II,but only partially inhibited CaM kinase I at a more than l0-fold higher concentration. Peptide 1 also inhibited a plant calcium dependent protein kinase (CDPK), whereas peptide 2 did not. The results suggested that the two peptides possibly have different effects on signal transduction pathways mediated by CaM and CDPK in plants.We also developed a transcriptional induction system in transgenic plants because it was thought to be required for the expression of the functionaI peptides in vivo. The system consists of a chimeric transcription factor (GVG) which acts as a strong transactivator only in the presence of glucocorticoid and a promoter recognized by GVG.In an experiment with a luciferase gene as a reporter, the system was found to work effectively in transgenic tobacco and Arabidopsis This system is expected to be useful for the expression of not only the mimicking pepddes but also other genes in transgenic plants.

0配基多肽可以根据其氨基酸序列模拟各种三维结构。在模拟多肽的思想基础上，我们尝试在体内模拟具有寡肽的非肽配体的功能。首先，我们通过修饰载体噬菌体M13mpl8构建了一个新的噬菌体随机多肽文库。对该噬菌体文库进行了钙调蛋白(CaM)结合肽的筛选，鉴定出两个寡肽(分别命名为WDTVR1SF和WPSLQAlR)。这两个多肽对CaM依赖的激酶I和II(CaM激酶I和Il)有不同的抑制作用。肽1抑制CaM激酶I，但不抑制CaM激酶II，而肽2抑制CaM激酶II，但在10倍以上的浓度下仅部分抑制CaM激酶I。多肽1也抑制植物钙依赖蛋白激酶(CDPK)，而多肽2不抑制。结果表明，这两种多肽在植物中CaM和CDPK介导的信号转导途径中可能具有不同的作用。我们还建立了转基因植物的转录诱导系统，因为这两种多肽被认为是体内表达功能多肽所必需的。该系统由一个仅在糖皮质激素存在时起反式激活作用的嵌合转录因子(GVG)和一个被GVG识别的启动子组成。在以荧光素酶基因为报告基因的实验中，该系统被发现在转基因烟草和拟南芥中有效地工作。该系统不仅可用于表达模拟肽，还可用于在转基因植物中表达其他基因。