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Page count: 180

Antibodies are a critical part of modern medicine, comprising 20% of the novel drugs approved by the FDA in 2015. While there are several platforms available to develop antibodies against a given antigen, they generally take months to carry out or require expensive equipment and extensive technical expertise. Recently, Robinson et al. demonstrated that SHuffle T7-Express cells are able to form disulfide bonds within the cytoplasm, enabling the cytoplasmic production of full-length antibodies. Using this technology, this work develops a novel antibody selection platform using chloramphenicol acetyltransferase (CAT) export to the periplasm via the Tat pathway. Within each cell two constructs are expressed within the cytoplasm, an antibody and the CAT-antigen fusion, which is rapidly exported into the periplasm resulting in chloramphenicol sensitivity. In cases where the expressed antibody displays sufficient binding activity with the antigen, the resulting complex will hinder the export of the CAT-antigen fusion, restoring chloramphenicol resistance. In this study we demonstrate that Tat enabled CAT export reliably reports the binding interaction between various antigens and their antibody cognates. We have further validated this approach by utilizing it to identify known antibody-antigen pairs from a mock library with 100-fold excess of non-specific pairs. Finally, one of the successful antibodyantigen pairs was used to construct a CDR-H3 library for screening with this platform and initial results appear promising. We believe that our platform will provide a simple and cost effective method for rapid discovery of clinically relevant antibodies for use with a wide range of diseases and illnesses.