Given the importance of protein folding to myriad biological applications, it is not surprising that several methods of monitoring protein folding and solubility have been engineered. However, while many reporters have been shown to be effective as reporters of folding behavior (e.g., solubility, aggregation, inclusion body formation), all report folding that occurs in the cytoplasm of E. coli. At present, there are no reports of cell-based assays for monitoring protein folding in extracytoplasmic compartments such as the periplasmic space. Accordingly, we report the development of a genetic selection for periplasmic protein solubility and folding in vivo using the signal recognition particle (SRP)-dependent pathway. The advantage conferred by use of the SRP-dependent pathway is that target proteins are extruded directly into the periplasm without residence time in the cytoplasmic folding environment. Using this system, we have characterized the in vivo periplasmic folding and solubility of several classes of proteins including amyloid-β peptides and single-chain Fv antibody fragments. In addition, we have demonstrated its potential for the directed evolution of proteins in the periplasm. Finally, by coupling this SRP-dependent folding assay with a previously developed cytoplasmic folding assay based on the Tat-dependent pathway, it is possible to observe, in an unbiased manner, the difference in the folding environments of the cytoplasmic and periplasmic cellular compartments. The application of this system is geared toward unbiased whole-cell screening of combinatorial libraries to determine the optimal folding environment for heterologously expressed proteins.

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