Lima Guilherme M, Atrazhev Alexey, Sarkar Susmita, Sojitra Mirat, Reddy Revathi, Torres-Obreque Karin, de Oliveira Rangel-Yagui Carlota, Macauley Matthew S, Monteiro Gisele, Derda Ratmir
Departamento de Tecnologia Bioquímico-Farmacêutica, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, 05508 000, Brazil.
Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
ACS Chem Biol. 2022 Nov 18;17(11):3024-3035. doi: 10.1021/acschembio.1c00835. Epub 2021 Dec 20.
Phage display links the phenotype of displayed polypeptides with the DNA sequence in the phage genome and offers a universal method for the discovery of proteins with novel properties. However, the display of large multisubunit proteins on phages remains a challenge. A majority of protein display systems are based on monovalent phagemid constructs, but methods for the robust display of multiple copies of large proteins are scarce. Here, we describe a DNA-encoded display of a ∼ 200 kDa tetrameric l-asparaginase protein on M13 and fd phages produced by ligation of SpyCatcher-Asparaginase fusion (ScA) and PEGylated-ScA (PEG-ScA) to barcoded phage clones displaying SpyTag peptide. Starting from the SpyTag display on p3 or p8 coat proteins yielded constructs with five copies of ScA displayed on p3 (ScA-p3), ∼100 copies of ScA on p8 protein (ScA-p8) and ∼300 copies of PEG-ScA on p8 protein (PEG-ScA-p8). Display constructs of different valencies and chemical modifications on protein (e.g., PEGylation) can be injected into mice and analyzed by deep sequencing of the DNA barcodes associated with phage clones. In these multiplexed studies, we observed a density and protein-dependent clearance rate . Our observations link the absence of PEGylation and increase in density of the displayed protein with the increased rate of the endocytosis by cells . In conclusion, we demonstrate that a multivalent display of l-asparaginase on phages could be used to study the circulation life of this protein , and such an approach opens the possibility to use DNA sequencing to investigate multiplexed libraries of other multisubunit proteins .
噬菌体展示将展示的多肽的表型与噬菌体基因组中的DNA序列联系起来，为发现具有新特性的蛋白质提供了一种通用方法。然而，在噬菌体上展示大型多亚基蛋白质仍然是一个挑战。大多数蛋白质展示系统基于单价噬菌粒构建体，但用于稳健展示大型蛋白质多拷贝的方法却很少。在这里，我们描述了一种通过将SpyCatcher-天冬酰胺酶融合体（ScA）和聚乙二醇化ScA（PEG-ScA）连接到展示SpyTag肽的条形码噬菌体克隆上，在M13和fd噬菌体上对约200 kDa的四聚体L-天冬酰胺酶蛋白进行DNA编码展示。从p3或p8外壳蛋白上展示SpyTag开始，得到了在p3上展示五个ScA拷贝的构建体（ScA-p3）、在p8蛋白上展示约100个ScA拷贝（ScA-p8）以及在p8蛋白上展示约300个PEG-ScA拷贝（PEG-ScA-p8）。不同价态和蛋白质化学修饰（如聚乙二醇化）的展示构建体可以注射到小鼠体内，并通过对与噬菌体克隆相关的DNA条形码进行深度测序来分析。在这些多重研究中，我们观察到了密度和蛋白质依赖性清除率。我们的观察结果表明，缺乏聚乙二醇化以及展示蛋白密度的增加与细胞内吞作用速率的增加有关。总之，我们证明了噬菌体上L-天冬酰胺酶的多价展示可用于研究该蛋白的循环寿命，并且这种方法为利用DNA测序研究其他多亚基蛋白质的多重文库开辟了可能性。
