Generation of protein lineages with new sequence spaces by functional salvage screen

A variety of different methods to generate diverse proteins, including random mutagenesis and recombination, are currently available and most of them accumulate the mutations on the target gene of a protein, whose sequence space remains unchanged. On the other hand, a pool of diverse genes, which is generated by random insertions, deletions and exchange of the homologous domains with different lengths in the target gene, would present the protein lineages resulting in new fitness landscapes. Here we report a method to generate a pool of protein variants with different sequence spaces by employing green fluorescent protein (GFP) as a model protein. This process, designated functional salvage screen (FSS), comprises the following procedures: a defective GFP template expressing no fluorescence is first constructed by genetically disrupting a predetermined region(s) of the protein and a library of GFP variants is generated from the defective template by incorporating the randomly fragmented genomic DNA from Escherichia coli into the defined region(s) of the target gene, followed by screening of the functionally salvaged, fluorescence-emitting GFPs. Two approaches, sequencedirected and PCR-coupled methods, were attempted to generate the library of GFP variants with new sequences derived from the genomic segments of E.coli. The functionally salvaged GFPs were selected and analyzed in terms of the sequence space and functional properties. The results demonstrate that the functional salvage process not only can be a simple and effective method to create protein lineages with new sequence spaces, but also can be useful in elucidating the involvement of a specific region(s) or domain(s) in the structure and function of protein.