Improving Protein Folding Efficiency by Directed Evolution Using the GFP Folding Reporter

AbstractRecombinant protein expression in heterologous hosts such as Escherichia coli (E. coli) can provide large amounts of a protein of interest. Often, expression can result in the accumulation of the recombinant protein as inactive, insoluble inclusion bodies (1). When attempts at refolding inclusion bodies fail (2,3), directed evolution methods can provide an alternative route to stable, correctly-folded proteins (4–6). In directed evolution methods, typically a library of genetic variants is screened for improved folding and solubility. When high-throughput function or activity screens are unavailable for the protein of interest, a folding reporter assay can be used (7). Folding reporter assays typically couple the folding of the test protein with that of a protein with an easily-detectable function, such as an antibiotic resistance protein or a fluorescent protein such as green fluorescent protein (GFP) (7). A cyclical process of DNA recombination, mutagenesis, and subsequent rescreening can produce variants with further improvement (4–7). The GFP method has been used to improve the folding of several proteins while preserving the test protein enzymatic function and native-like structure (7–10). KeywordsGreen Fluorescent ProteinGreen Fluorescent Protein FusionGreen Fluorescent Protein Fusion ProteinCorrect InsertMaster PlateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.