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Protein Engineering vol. 16 no. 12 pp. 1099-1105, 2003
© 2003 Oxford University Press

Functional tuning of a salvaged green fluorescent protein variant with a new sequence space by directed evolution

Sung-Hun Nam1, Ki-Hoon Oh2, Geun-Joong Kim3,4 and Hak-Sung Kim1,4

1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusung-dong, Yusung-gu, Taejon, 305-701, 2R&D Center of Bioproducts, Institute of Science and Technology, CJ Corp., 522-1, Dokpyong-ri, Majang-myon, Ichon, 467-810 and 3Institute of Biotechnological Industry, College of Engineering, Inha University, 253, Yonghyun-dong, Nam-gu, Incheon, 402-751, Korea

4 To whom correspondence should be addressed. e-mail: hskim{at}mail.kaist.ac.krorgeunkim@inha.ac.kr

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 ~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.

Received March 31, 2003; revised October 30, 2003; accepted October 31, 2003


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