Functional tuning of a salvaged green fluorescent protein variant with a new sequence space by directed evolution.
نویسندگان
چکیده
We previously reported a method, designated functional salvage screen (FSS), to generate protein lineages with new sequence spaces through the functional or structural salvage of a defective protein by employing green fluorescent protein (GFP) as a model protein. Here, in an attempt to mimic a step in the natural evolution process of proteins, the functionally salvaged mutant GFP-I5 with new sequence space, but showing low fluorescence intensity and stability, was selected and fine-tuned by directed evolution. During a course of functional tuning, GFP-I5 was found to evolve rapidly, recovering the spectral traits to those of the parent GFPuv. The mutant 3E4 from the third round of directed evolution possessed four substitutions; three (F64L, E111V and K166Q) were at the original GFP gene and the other (K8N) at the inserted segment. The fluorescence intensity of 3E4 was approximately 28-fold stronger than GFP-I5, and other spectral properties were retained. Biochemical and biophysical investigations suggested that the fine-tuning by directed evolution led the salvaged variant GFP-I5 to a functionally favorable structure, resulting in recovery of stability and fluorescence. Site-directed mutagenesis of the mutated amino acid residues in both GFPuv and GFP-I5 revealed that each amino acid residue has a different effect on the fluorescence intensity, which implies that 3E4 adopted a new evolutionary path with respect to fluorescence characteristics compared with the parent GFPuv. Directed evolution in conjunction with FSS is expected to be used for generating protein lineages with new fitness landscapes.
منابع مشابه
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 ...
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عنوان ژورنال:
- Protein engineering
دوره 16 12 شماره
صفحات -
تاریخ انتشار 2003