Rentero Rebollo Inmaculada, Sabisz Michal, Baeriswyl Vanessa, Heinis Christian
Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
Nucleic Acids Res. 2014 Dec 16;42(22):e169. doi: 10.1093/nar/gku940. Epub 2014 Oct 27.
High-throughput sequencing was previously applied to phage-selected peptides in order to gain insight into the abundance and diversity of isolated peptides. Herein we developed a procedure to efficiently compare the sequences of large numbers of phage-selected peptides for the purpose of identifying target-binding peptide motifs. We applied the procedure to analyze bicyclic peptides isolated against five different protein targets: sortase A, urokinase-type plasminogen activator, coagulation factor XII, plasma kallikrein and streptavidin. We optimized sequence data filters to reduce biases originating from the sequencing method and developed sequence correction algorithms to prevent identification of false consensus motifs. With our strategy, we were able to identify rare target-binding peptide motifs, as well as to define more precisely consensus sequences and sub-groups of consensus sequences. This information is valuable to choose peptide leads for drug development and it facilitates identification of epitopes. We furthermore show that binding motifs can be identified after a single round of phage selection. Such a selection regimen reduces propagation-related bias and may facilitate application of phage display in non-specialized laboratories, as procedures such as bacterial infection, phage propagation and purification are not required.
高通量测序先前已应用于噬菌体选择的肽段，以深入了解分离肽段的丰度和多样性。在此，我们开发了一种程序，用于高效比较大量噬菌体选择的肽段的序列，以识别靶标结合肽基序。我们应用该程序分析针对五种不同蛋白质靶标的双环肽：分选酶A、尿激酶型纤溶酶原激活剂、凝血因子XII、血浆激肽释放酶和链霉亲和素。我们优化了序列数据过滤器，以减少源自测序方法的偏差，并开发了序列校正算法，以防止识别错误的共有基序。通过我们的策略，我们能够识别罕见的靶标结合肽基序，并更精确地定义共有序列和共有序列亚组。这些信息对于选择用于药物开发的肽先导物很有价值，并且有助于确定表位。我们还表明，在一轮噬菌体选择后即可识别结合基序。这种选择方案减少了与繁殖相关的偏差，并且可能有助于噬菌体展示在非专业实验室中的应用，因为不需要诸如细菌感染、噬菌体繁殖和纯化等程序。
