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  1. Abstract

    Here we describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineeredEscherichia colicells. 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.

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  2. Abstract

    Single‐chain variable fragment (scFv) antibodies have great potential for a range of applications including as diagnostic and therapeutic agents. However, production of scFvs is challenging because proper folding and activity depend on the formation of two intrachain disulfide bonds that do not readily form in the cytoplasm of living cells. Functional expression in bacteria therefore involves targeting to the more oxidizing periplasm, but yields in this compartment can be limiting due to secretion bottlenecks and the relatively small volume compared to the cytoplasm. In the present study, we evaluated an anti‐HER2 scFv, which is specific for human epidermal growth receptor 2 (HER2) overexpressed in breast cancer, for functional expression in the cytoplasm ofEscherichia colistrains BL21(DE3) and SHuffle T7 Express, the latter of which is genetically engineered for cytoplasmic disulfide bond formation. Specifically, we observed much greater solubility and binding activity with SHuffle T7 Express cells, which likely resulted from the more oxidative cytoplasm in this strain background. We also found that SHuffle T7 Express cells were capable of supporting high‐level soluble production of anti‐HER2 scFvs with intact disulfide bonds independent of variable domain orientation, providing further evidence that SHuffle T7 Express is a promising host for laboratory and preparative expression of functional scFv antibodies.

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