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

Autor(es)

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

• Tipo de documento: Artigo Científico
• Fonte: ACS Chemical Biology; v. 17, n. 11, p. 12-pg., 2022-11-18.

Resumo

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 similar to 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 (ScAp3), similar to 100 copies of ScA on p8 protein (ScA-p8) and similar to 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. (AU)

• Processo FAPESP: 19/09354-6 - Evolução dirigida da L-asparaginase para aprimoramento de sua estabilidade in vivo
• Beneficiário: Guilherme Meira Lima
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Mestrado
• Processo FAPESP: 16/22065-5 - Pegilação N-terminal de proteínas e purificação por sistemas aquosos bifásicos
• Beneficiário: Carlota de Oliveira Rangel Yagui
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 18/15041-8 - Expressão de L-asparaginase de Erwinia chrysanthemi em tecnologia de síntese proteica livre de células
• Beneficiário: Guilherme Meira Lima
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 13/08617-7 - Produção de L-asparaginase extracelular: da bioprospecção à engenharia de um biofármaco antileucêmico
• Beneficiário: Adalberto Pessoa Junior
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 18/15104-0 - Ensaios pré-clínicos de proteoformas de asparaginase glicoproteicas ou resistentes a proteases séricas.
• Beneficiário: Gisele Monteiro
• Modalidade de apoio: Auxílio à Pesquisa - Regular