Site-specific incorporation of unnatural amino acids into urate oxidase in Escherichia coli.
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.
Chen
Mingjie
M
School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, People's Republic of China.
Cai
Lei
L
Fang
Zhengzhi
Z
Tian
Hong
H
Gao
Xiangdong
X
Yao
Wenbing
W
D019943:Amino Acid Substitution / Q000235:genetics*; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D001386:Azides / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000378:metabolism; D014503:Urate Oxidase / Q000737:chemistry* / Q000235:genetics
D000596:Amino Acids; D001386:Azides; D012346:RNA, Transfer, Amino Acyl; C027162:4-azidophenylalanine; D010649:Phenylalanine; D014503:Urate Oxidase; D000604:Amino Acyl-tRNA Synthetases