Synthesis and sequence optimization of GFP mutants containing aromatic non-natural amino acids at the Tyr66 position.
In order to alter the fluorescence properties of green fluorescent protein (GFP), aromatic non-natural amino acids were introduced into the Tyr66 position of GFP in a cell-free translation system using a four-base codon method. Two non-natural mutants (O-methyltyrosine and p-aminophenylalanine mutants) out of 18 mutants showed blue-shifted but weak fluorescence compared with wild-type GFP. Then the aminophenylalanine mutant was sequence optimized by introducing random mutations around the Tyr66 site. For this purpose, a method for random mutation of non-natural proteins in a cell-free system was developed. Three aminophenylalanine mutants with Y145F, Y145L and Y145 M mutations were obtained, which exhibited increased fluorescence by 1.5-, 3- and 4-fold, respectively. These results indicate that random mutation around non-natural amino acids is useful strategy in order to improve protein functions that are reduced by non-natural amino acid incorporation. The method described here will be applicable to other non-natural mutant proteins in a high-throughput manner.
Kajihara
Daisuke
D
Department of Bioscience and Biotechnology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.
Hohsaka
Takahiro
T
Sisido
Masahiko
M
D002474:Cell-Free System; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D012343:RNA, Transfer / Q000235:genetics*; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics*
D003062:Codon; D049452:Green Fluorescent Proteins; C009722:4-aminophenylalanine; D014443:Tyrosine; D010649:Phenylalanine; D012343:RNA, Transfer