Rapid protein-folding assay using green fluorescent protein

Formation of the chromophore of green fluorescent protein (GFP) depends on the correct folding of the protein. We constructed a "folding reporter" vec tor, in which a test protein is expressed as an N-terminal fusion with GFP. Using a test panel of 20 proteins, we demonstrated that the fluorescence o f Escherichia coli cells expressing such GFP fusions is related to the prod uctive folding of the upstream protein domains expressed alone. We used thi s fluorescent indicator of protein folding to evolve proteins that are norm ally prone to aggregation during expression in E. coli into closely related proteins that ford robustly and are fully soluble and functional. This app roach to improving protein folding does not require functional assays for t he protein of interest and provides a simple route to improving protein fol ding and expression by directed evolution.