Department of Clinical Physiology and Nuclear Medicine & Cluster for Molecular Imaging, Copenhagen University Hospital-Rigshospitalet & Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
Molecules. 2023 Jul 10;28(14):5318. doi: 10.3390/molecules28145318.
The larger size and diversity of phage display peptide libraries enhance the probability of finding clinically valuable ligands. A simple way of increasing the throughput of selection is to mix multiple peptide libraries with different characteristics of displayed peptides and use it as biopanning input. In phage display, the peptide is genetically coupled with a biological entity (the phage), and the representation of peptides in the selection system is dependent on the propagation capacity of phages. Little is known about how the characteristics of displayed peptides affect the propagation capacity of the pooled library. In this work, next-generation sequencing (NGS) was used to investigate the amplification capacity of three widely used commercial phage display peptide libraries (Ph.D.™-7, Ph.D.™-12, and Ph.D.™-C7C from New England Biolabs). The three libraries were pooled and subjected to competitive propagation, and the proportion of each library in the pool was quantitated at two time points during propagation. The results of the inter-library competitive propagation assay led to the conclusion that the propagation capacity of phage libraries on a population level is decreased with increasing length and cyclic conformation of displayed peptides. Moreover, the enrichment factor (EF) analysis of the phage population revealed a higher propagation capacity of the Ph.D.-7 library. Our findings provide evidence for the contribution of the length and structural conformation of displayed peptides to the unequal propagation rates of phage display libraries and suggest that it is important to take peptide characteristics into account once pooling multiple combinatorial libraries for phage display selection through biopanning.
噬菌体展示肽文库的较大规模和多样性提高了发现具有临床价值配体的概率。增加选择通量的一种简单方法是将具有不同展示肽特性的多个肽文库混合，并将其用作生物淘选输入。在噬菌体展示中，肽与生物实体（噬菌体）在遗传上偶联，并且选择系统中肽的表示取决于噬菌体的繁殖能力。关于展示肽的特性如何影响汇集文库的繁殖能力，人们知之甚少。在这项工作中，使用下一代测序（NGS）来研究三种广泛使用的商业噬菌体展示肽文库（来自 New England Biolabs 的 Ph.D.™-7、Ph.D.™-12 和 Ph.D.™-C7C）的扩增能力。将这三个文库混合并进行竞争繁殖，并在繁殖过程中的两个时间点定量测定库中每个文库的比例。库间竞争繁殖测定的结果得出结论，噬菌体文库在群体水平上的繁殖能力随着展示肽长度和环状构象的增加而降低。此外，噬菌体群体的富集因子（EF）分析显示 Ph.D.-7 文库具有更高的繁殖能力。我们的研究结果为展示肽的长度和结构构象对噬菌体展示文库的不等繁殖率的贡献提供了证据，并表明一旦通过生物淘选对多个组合文库进行噬菌体展示选择进行混合，就必须考虑肽特性。
