A Tunable Bacterial Selection System for Engineering Condition-Dependent Protein Function

Protein engineering towards the development of protein switches requires evolution in opposing directions, producing proteins that are highly active in one state and inactive in the other. Numerous assays exist to aid in evolving protein function, however these assays are of limited use in the development of condition-dependent protein function, as they do not control for absence of function in the off-state. To enable bi-directional evolution of conditional function, we have engineered an in vivo assay with a selection parameter that can be tuned, allowing for enrichment of the on- or off-state as required by external conditions. This assay combines principles from two previously established in vivo assays: the â??hitchhikerâ?? assay¹, a selection assay for protein-protein interactions based on the production of beta-lactamase; and the â??band-passâ?? assay², an externally tuneable bacterial selection system for beta-lactamase activity. The resulting genetic circuit enables one to select for cells with specific levels of beta-lactamase activity, which correspond to different protein-protein interaction strengths. We demonstrated the function of this assay using designed leucine zipper peptides with a range of interaction strengths, comparing the results with data from in vitro characterization of the peptides. We will use this assay in the development of a synthetic photo-controlled protein, using the ability of the assay to select for cells with the protein switch in the active or inactive state, depending on the experimental set up. It is anticipated that this assay could be used to facilitate the directed evolution of a wide variety of protein switches involving protein-protein interactions