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Adding New Properties to Proteins via Reassigning AGA Codon to Encode for Unnatural Amino Acids in E. coli
Author(s) -
Wang Yiyan,
Tsao Meng-Lin
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.lb113
Subject(s) - genetic code , amino acid , transfer rna , stop codon , genetics , mutant , synonymous substitution , biology , escherichia coli , codon usage bias , gene , biochemistry , chemistry , computational biology , rna , genome
The ability to introduce unnatural amino acids ( uaa s) with novel chemical, physical and biological properties at specific sites into proteins adds a new dimension to study protein structure and function. The UAG amber stop codon has been utilized as the top choice to encode for uaa s. To provide more powerful codons to further expand the genetic code, here we report our study in reassignment of AGA codon, one of the rare codons, from arginine to uaa s in E. coli . This was achieved through a two‐step approach. Firstly, the anticodon recognizing region of Methanococcus Jannaschii tyrosyl‐tRNA synthetase ( Mj TyrRS) was altered to host tRNA UCU instead of original tRNA GUA by using a mutant GFP uv as the reporter. Secondly, subsequently modifications on amino acid binding pocket of the mutated MjTyr RS uaa allowed the incorporation of target unnatural amino acids into proteins in response to AGA codon. Our results showed that in E.coli the suppression rate of AGA codon by the evolved MjTyr RS uaa ‐ Mj tRNA UCU pair was generally above 90% and the uaa ‐decorated protein yield reached about 100 mg/L. This research provided a powerful new approach to add new building blocks to the genetic code and to the structural/functional study of protein with novel properties. The project was sponsored by UC Merced start‐up fund.