Full-length monoclonal antibodies (mAbs) are indispensable tools in many research, diagnostic, and clinical applications. However, developing specific full-length mAbs against novel targets with existing technologies remains a challenge, as typically they require a series of laborious steps, expensive materials, or purification of the target antigen. In previous work, we achieved efficient production of soluble, full-length immunoglobulin G (IgG) antibodies within the cytoplasm of an engineered E. coli strain, SHuffle. Here, we adapt this system for the isolation of specific full-length IgGs within living E. coli cells by implementing a version of the protein fragment complementation assay (PCA). This was achieved by genetically fusing two inactive fragments of enolpyruvylshikimate-3-phosphate synthase (AroA), an enzyme essential for bacterial viability on minimal media, to a full-length IgG and its antigen. Upon expressing these constructs in the cytoplasm of SHuffle cells lacking the aroA gene (SHuffle delta aroA), the binding interaction between the IgG and its antigen facilitates reassembly of functional AroA thereby restoring cell growth on minimal media. We show that the AroA PCA reliably reports the specific binding of a full-length antibody against a target antigen, namely E. coli maltose binding protein (MBP), within the cytosol of SHuffle delta aroA cells. This approach was further validated by employing it as a reporter for intracellular binding of several other well-characterized antibody-antigen pairs. Finally, we apply the AroA PCA system to the isolation of a known IgG-antigen pair from a mock library containing an excess of non-specific IgG-antigen pairs in addition to the cognate pair, thereby demonstrating the potency of our IgG isolation technology. We anticipate that our method will facilitate discovery and engineering of entirely new antibodies in a single step and may provide a simple platform for rapidly selecting full-length IgGs for use in a wide range of applications.

Functional selection of full-length antibodies in the cytoplasm of living bacterial cells

251st National Meeting (2016)

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