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Published online before print July 2, 2008
Protein Science, DOI: 10.1110/ps.034587.108
 Copyright © 2008 The Protein Society
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Site-Specific Incorporation of Unnatural Amino Acids into Urate oxidase in Escherichia coli

Mingjie Chen, Lei Cai, Zhengzhi Fang, Hong Tian, Xiangdong Gao, and Wenbing Yao1

China Pharmaceutical University

(RECEIVED January 18, 2008; ACCEPTED June 25, 2008)

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 Phe170 or Phe281 in urate oxidase. The products were purified and their enzyme activities were analyzed. In addition, we optimized the system by adding an "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.

Keywords: Stability and mutagenesis; Enzymes; Active sites; Isolation, Characterization, Chromatography; Affinity/immuno methods; cDNA, Mutagenesis, Cloning; Mutagenesis (site-directed and general); Expression systems

1 E-mail: wbyao{at}cpu.edu.cn


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