NDLI: Experimental Evolution of a Green Fluorescent Protein Composed of 19 Unique Amino Acids without Tryptophan

Content Provider	Springer Nature Link
Author	Kawahara Kobayashi, Akio Hitotsuyanagi, Mitsuhiro Amikura, Kazuaki Kiga, Daisuke
Copyright Year	2014
Abstract	At some stage of evolution, genes of organisms may have encoded proteins that were synthesized using fewer than 20 unique amino acids. Similar to evolution of the natural 19-amino-acid proteins GroEL/ES, proteins composed of 19 unique amino acids would have been able to evolve by accumulating beneficial mutations within the 19-amino-acid repertoire encoded in an ancestral genetic code. Because Trp is thought to be the last amino acid included in the canonical 20-amino-acid repertoire, this late stage of protein evolution could be mimicked by experimental evolution of 19-amino-acid proteins without tryptophan (Trp). To further understand the evolution of proteins, we tried to mimic the evolution of a 19-amino-acid protein involving the accumulation of beneficial mutations using directed evolution by random mutagenesis on the whole targeted gene sequence. We created active 19-amino-acid green fluorescent proteins (GFPs) without Trp from a poorly fluorescent 19-amino-acid mutant, S1-W57F, by using directed evolution with two rounds of mutagenesis and selection. The N105I and S205T mutations showed beneficial effects on the S1-W57F mutant. When these two mutations were combined on S1-W57F, we observed an additive effect on the fluorescence intensity. In contrast, these mutations showed no clear improvement individually or in combination on GFPS1, which is the parental GFP mutant composed of 20 amino acids. Our results provide an additional example for the experimental evolution of 19-amino-acid proteins without Trp, and would help understand the mechanisms underlying the evolution of 19-amino-acid proteins. (236 words)
Starting Page	75
Ending Page	86
Page Count	12
File Format	PDF
ISSN	01696149
Journal	Origins of Life and Evolution of Biospheres
Volume Number	44
Issue Number	2
e-ISSN	15730875
Language	English
Publisher	Springer Netherlands
Publisher Date	2014-11-16
Publisher Place	Dordrecht
Access Restriction	Subscribed
Subject Keyword	Directed evolution Green fluorescent protein Tryptophan 19 amino acids Genetic code Life Sciences Astrophysics and Astroparticles Earth Sciences Astronomy, Observations and Techniques Biochemistry
Content Type	Text
Resource Type	Article
Subject	Medicine Ecology, Evolution, Behavior and Systematics Space and Planetary Science

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