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Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli

Here we describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered Escherichia coli cells. The method is based on the transport of a bifunctional substrate comprised of an antigen fused to chlora...

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Detalles Bibliográficos
Autores principales: Robinson, Michael-Paul, Jung, Jinjoo, Lopez-Barbosa, Natalia, Chang, Matthew, Li, Mingji, Jaroentomeechai, Thapakorn, Cox, Emily C., Zheng, Xiaolu, Berkmen, Mehmet, DeLisa, Matthew P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10267130/
https://www.ncbi.nlm.nih.gov/pubmed/37316535
http://dx.doi.org/10.1038/s41467-023-39178-x
Descripción
Sumario:Here we describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered Escherichia coli cells. The method is based on the transport of a bifunctional substrate comprised of an antigen fused to chloramphenicol acetyltransferase, which allows positive selection of bacterial cells co-expressing cytoplasmic IgGs called cyclonals that specifically capture the chimeric antigen and sequester the antibiotic resistance marker in the cytoplasm. The utility of this approach is first demonstrated by isolating affinity-matured cyclonal variants that specifically bind their cognate antigen, the leucine zipper domain of a yeast transcriptional activator, with subnanomolar affinities, which represent a ~20-fold improvement over the parental IgG. We then use the genetic assay to discover antigen-specific cyclonals from a naïve human antibody repertoire, leading to the identification of lead IgG candidates with affinity and specificity for an influenza hemagglutinin-derived peptide antigen.