DNA-Encoded Multivalent Display of Chemically Modified Protein Tetramers on Phage: Synthesis and in Vivo Applications.

Guilherme M Lima, Alexey Atrazhev, Susmita Sarkar, Mirat Sojitra, Revathi Reddy, Karin Torres-Obreque, Carlota de Oliveira Rangel-Yagui, Matthew S Macauley, Gisele Monteiro, Ratmir Derda

ACS chemical biology 2022 Nov 18

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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 in vivo. 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 vivo. In conclusion, we demonstrate that a multivalent display of l-asparaginase on phages could be used to study the circulation life of this protein in vivo, and such an approach opens the possibility to use DNA sequencing to investigate multiplexed libraries of other multisubunit proteins in vivo.

Citation

Guilherme M Lima, Alexey Atrazhev, Susmita Sarkar, Mirat Sojitra, Revathi Reddy, Karin Torres-Obreque, Carlota de Oliveira Rangel-Yagui, Matthew S Macauley, Gisele Monteiro, Ratmir Derda. DNA-Encoded Multivalent Display of Chemically Modified Protein Tetramers on Phage: Synthesis and in Vivo Applications. ACS chemical biology. 2022 Nov 18;17(11):3024-3035