Fine tuning of functionally salvaged green fluorescent protein variant by directed evolution

Abstract

Functional salvage screen (FSS) method was invented as a novel method that could create new protein lineage with different sequence space. Functionally salvaged GFP variants with randomly inserted fragments were generated by FSS. However, these mutants showed little fluorescence intensity. Among these variants, GFP-I5 was selected and directly evolved to optimize the spectral property. As a result, best mutant, E-2S4 was obtained from the library generated by ep-PCR and DNA shuffling. It possessed the mutations in F64L, E111V, K166Q and Kins-8N residues. The fluorescence intensity of purified protein of E-2S4 was ~28-fold improved over that of GFP-I5, but other spectral properties were unchanged. This improvement of fluorescence was also observed in the whole cell of E.coli. Purified protein of E-2S4 has different conformation with other GFP mutants. In order to investigate the effect of each mutated residues on fluorescence intensity, site-directed mutagenesis was carried out. Site-specific mutations in other templates showed the different effect of fluorescence intensity. These results suggest that salvaged variant, GFP-I5 had new sequence space by the incorporation of 12 amino acids and it was subjected to new evolution pathway in further artificial evolution.