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Site‐specific incorporation of unnatural amino acids into urate oxidase in Escherichia coli
Author(s) -
Chen Mingjie,
Cai Lei,
Fang Zhengzhi,
Tian Hong,
Gao Xiangdong,
Yao Wenbing
Publication year - 2008
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.034587.108
Subject(s) - urate oxidase , allantoin , biochemistry , chemistry , escherichia coli , oxidase test , enzyme , uric acid , phenylalanine , aminoacyl trna synthetase , amino acid , amino acyl trna synthetases , transfer rna , gene , rna
Urate oxidase catalyzes the oxidation of uric acid with poor solubility to produce 5‐hydroxyisourate and allantoin. Since allantoin is excreted in vivo, urate oxidase has the potential to be a therapeutic target for the treatment of gout. However, its severe immunogenicity limits its clinical application. Furthermore, studies on the structure–function relationships of urate oxidase have proven difficult. We developed a method for genetically incorporating p ‐azido‐L‐phenylalanine into target protein in Escherichia coli in a site‐specific manner utilizing a tyrosyl suppressor tRNA/aminoacyl‐tRNA synthetase system. We substituted p ‐azido‐L‐phenylalanine for Phe 170 or Phe 281 in urate oxidase. The products were purified and their enzyme activities were analyzed. In addition, we optimized the system by adding a “Shine‐Dalgarno (SD) sequence” and tandem suppressor tRNA. This method has the benefit of site‐specifically modifying urate oxidase with homogeneous glycosyl and PEG derivates, which can provide new insights into structure–function relationships and improve pharmacological properties of urate oxidase.