Department of Chemical Engineering, University of California, Santa Barbara, CA 93106, USA.
Protein Eng Des Sel. 2010 Jan;23(1):9-17. doi: 10.1093/protein/gzp065.
A quantitative screening method was developed to enable isolation and affinity maturation of peptide ligands specific for a given target from peptide libraries displayed on the outer surface of Escherichia coli using multi-parameter flow cytometry. From a large, random 15-mer peptide library, screening identified a core motif of W-E/D-W-E/D that conferred binding to vascular endothelial growth factor (VEGF). One cycle of affinity maturation resulted in the identification of several families of VEGF-binding peptides having distinct consensus sequences, from which a preferred disulfide constraint emerged. In the second affinity maturation cycle, high affinity peptides were favored by the addition of a decoy protein that bound an adjacent epitope on the display scaffold. The decoy apparently reduced rebinding or avidity effects, and the resulting peptides exhibited consensus at 12 of 19 amino acid positions. Peptides identified and affinity matured using bacterial display were remarkably similar to the best affinity matured using phage display and exhibited comparable dissociation constants (within 2-fold; K(D) = 4.7 x 10(-7) M). Screening of bacterial-displayed peptide libraries using cytometry enabled optimization of screening conditions to favor affinity and specificity and rapid clonal characterization. Bacterial display thus provides a new quantitative tool for the discovery and evolutionary optimization of protein-specific peptide ligands.
建立了一种定量筛选方法，能够从在大肠杆菌外表面展示的肽文库中筛选出针对特定靶标的肽配体，并使用多参数流式细胞术进行亲和成熟。从一个大型的、随机的 15 肽文库中，筛选出了一个与血管内皮生长因子（VEGF）结合的 W-E/D-W-E/D 核心基序。一轮亲和成熟后，鉴定出了几个具有不同共有序列的 VEGF 结合肽家族，从中出现了一个优选的二硫键约束。在第二轮亲和成熟循环中，通过添加与展示支架上相邻表位结合的诱饵蛋白，有利于高亲和力肽的选择。诱饵蛋白显然减少了再结合或亲和力效应，而得到的肽在 19 个氨基酸位置中的 12 个位置上具有共有序列。使用细菌展示鉴定和亲和成熟的肽与使用噬菌体展示鉴定和亲和成熟的肽非常相似，且解离常数相当（相差 2 倍以内；K(D) = 4.7 x 10(-7) M）。使用流式细胞术筛选细菌展示的肽文库，可以优化筛选条件，有利于亲和力和特异性，并可以快速进行克隆表征。因此，细菌展示为发现和进化优化蛋白质特异性肽配体提供了一种新的定量工具。
