Screening and Application of Ligand-mimicking Peptides

脂肽可以模拟不同的三维结构，这取决于它们的氨基酸序列。基于模拟多肽的思想，我们尝试在体内用寡肽模拟非肽配体的功能。首先，我们通过修饰噬菌体载体Ml3mpl8构建了新的噬菌体随机肽文库。对该噬菌体文库进行钙调素（CaM）结合肽筛选，鉴定出两个寡肽（分别称为肽1和肽2；WDTVRlSF和WPSLQAlR）。这两种多肽对CaM依赖性激酶I和II （CaM激酶I和Il）有不同的抑制作用。肽1抑制CaM kinase I，但不抑制CaM kinase II，而肽2抑制CaM kinase II，但仅部分抑制CaM kinase I，其浓度高于10倍以上。肽1也抑制植物钙依赖性蛋白激酶（CDPK），而肽2则没有。结果表明，这两种多肽可能对植物中CaM和CDPK介导的信号转导途径有不同的影响。我们还在转基因植物中开发了一个转录诱导系统，因为它被认为是在体内表达功能肽所必需的。该系统由一个嵌合转录因子（GVG）和一个被GVG识别的启动子组成，GVG仅在糖皮质激素存在时作为强反激活因子。在以荧光素酶基因为报告基因的实验中，发现该系统在转基因烟草和拟南芥中有效地工作，该系统不仅可用于转基因植物中模拟肽的表达，还可用于其他基因的表达。

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.