Previously, we have developed and characterized a genetic selection for protein folding and solubility in living bacterial cells based on the inherent quality control mechanism of the bacterial twin-arginine translocation (Tat) pathway. We have extended this selection strategy to isolate recombinatorial stable single-chain immunotherapeutics and de novo proteins, in the reducing cytoplasm of E. coli. This is significant as members of the former class of recombinant proteins (e.g., single chain Fv antibody fragments, single chain T cell receptor ligands) are typically unstable when expressed in the reducing environment of the bacterial cytoplasm. Members of the latter class of proteins are notoriously difficult to screen for structure or function. We will discuss our recent efforts to characterize and employ this selection for directed evolution. In addition, we will discuss our efforts to reveal characteristics of protein quality control that have thus far remained elusive. By allowing the bacterial Tat pathway to exert folding quality control on expressed target protein sequences, we have generated a powerful tool for molecular engineering soluble proteins with great clinical and scientific promise.

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