Choi S J, Ahn M, Lee J S, Jung W J
Department of Chemistry, Kangnung National University, Korea.
Mol Cells. 1997 Oct 31;7(5):575-81.
Although the phage-displayed peptide libraries have been widely used for the biopanning of peptide specific for various types of target molecules, the selected peptides often have affinities too low for practical purposes. In this report, selection of a high affinity peptide ligand specific for human angiogenin is described. We constructed a random dodecapeptide library displayed on the gene III protein of filamentous bacteriophage by use of a phagemid. The peptide insert was flanked by two cysteines to constrain the peptide structure by a disulfide bond. Phages were collected from 1.5 x 10(3) independent transformants. After the library phages were allowed to bind to the human angiogenin coated on a plate, the phages bound to the actin-binding site of angiogenin were selectively eluted with actin. The activity of each phage clone was estimated and three high affinity phage clones were identified. The peptides displayed by the three phage clones were synthesized as fusion proteins with Escherichia coli maltose-binding protein, and used for the quantitative estimation of their affinities. By this procedure, we were able to select a peptide having a dissociation constant of about 60 nM.
尽管噬菌体展示肽库已被广泛用于针对各种类型靶分子的肽的生物淘选，但所选择的肽的亲和力通常因实际应用目的而过低。在本报告中，描述了针对人血管生成素的高亲和力肽配体的筛选。我们使用噬菌粒构建了一个展示在丝状噬菌体基因III蛋白上的随机十二肽库。肽插入片段两侧有两个半胱氨酸，通过二硫键来限制肽的结构。从1.5×10³个独立转化体中收集噬菌体。在库噬菌体与包被在平板上的人血管生成素结合后，用肌动蛋白选择性洗脱与血管生成素肌动蛋白结合位点结合的噬菌体。估计每个噬菌体克隆的活性，并鉴定出三个高亲和力噬菌体克隆。将这三个噬菌体克隆展示的肽合成为与大肠杆菌麦芽糖结合蛋白的融合蛋白，并用于定量估计它们的亲和力。通过这个程序，我们能够筛选出一种解离常数约为60 nM的肽。
