Protein-protein interactions are crucial to many cellular and industrial processes, including enzymatic pathways, molecule secretion, glycosylation, therapeutic production and function, and antibody function. Currently, many methods characterize protein interaction affinities in vitro and in vivo in the cytoplasm of various organisms, but to our knowledge, no such systems report folding and interaction of proteins in the periplasm. The periplasm of gram-negative bacteria offers an environment with different, beneficial characteristics for the production of heterologous (e.g. therapeutic) proteins. Thus, a means to easily and accurately detect and engineer protein interactions in the periplasm would be transformative for the development of novel antibodies and protein therapeutics. We have used a split β-lactamase protein complementation assay to successfully report interactions of several known interacting domains in the periplasm of Escherichia coli. Currently, we are using this technique to select for novel interactions in vivo from naive directed evolution libraries. This powerful methodology allows for a broad expansion of the protein engineer's toolbox for periplasmic expression and works towards a novel, efficient way to engineer new protein interactions in E. coli.
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