Reporters of protein folding and solubility deliver valuable insight on the in vivo protein folding process and allow for the engineering of proteins with improved solubility. Currently, many folding reporters exist that report folding behavior in the E. coli cytoplasm, but to our knowledge, no such systems have been shown to report folding of proteins in the periplasm. The periplasm is an important compartment of gram-negative bacteria where several unique post-translational processes contribute to protein solubility including disulfide bond formation and, more recently, engineered protein glycosylation. Using the recently discovered SRP transport pathway and a C-terminal fusion to TEM-1 Beta-lactamase, we have developed a reliable reporter of (1) intrinsic protein folding properties and (2) the effects of chaperone activity, disulfide bond formation, and glycosylation on folding and solubility of many proteins, including N-linked glycoproteins native to Campylobacter jejuni. In addition to reporting properties of proteins and their folding environment, we show that this technique can be used to engineer proteins with improved solubility via directed evolution.

Furthermore, we have expanded this technique to develop an assay for protein-protein interactions in the periplasm. Using a split-β-lactamase reporter system, we have successfully reported interactions of several known interacting domains. Currently, we are using this technique to select for novel interactions in vivo from naive directed evolution libraries. The combination of these two powerful methodologies allows for a broad expansion of the protein engineer's toolbox for periplasmic expression and ultimately works towards systems for engineering protein glycosylation in E. coli.

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