title: Designing and engineering of a site-specific incorporation of a keto group in uricase.
abstract: Urate oxidase is a potential therapeutic protein in the prevention and treatment of tumor lysis syndrome and hyperuricemia. However, its severe immunogenicity limits its clinical application. In our work, several strides have been made toward engineering site-specific modifications of keto groups in urate oxidase by using evolved Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s)/suppressor tRNA pairs to reduce its antigenicity. Our approach, described here, consisted of designing a M. jannaschii tyrosyl-tRNA synthetase library based on the homology modeling and molecular docking model of the species-specific TyrRS-Tyr complex. The active mutation was picked, and pBR-RS series vectors were constructed to define the relationship between the expression of aaRS and the efficiency of the orthogonal amber suppressor tRNA/synthetase system. Two sites based on the 3D structure of the Candida utilis uricase, Lys21 and Lys248, were substituted for p-acetyl-l-phenylalanine, and the yields were optimized. The products were purified, and their enzyme activities and antigenic properties were analyzed. The mutated uricase exhibited decreased antigenic properties, while its catalytic activities remained unchanged. This method imparts new insights into structure-function relationship research and provides a means by which site-specific modifications may be achieved by using PEG derivates to improve pharmacological properties of urate oxidase.
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, China.
authors.forename: Zhengzhi
authors.initials: Z
authors.lastname: Fang
authors.forename: Yaguang
authors.initials: Y
authors.lastname: Liu
authors.forename: Jingxian
authors.initials: J
authors.lastname: Liu
authors.forename: Renhua
authors.initials: R
authors.lastname: Sun
authors.forename: Hai
authors.initials: H
authors.lastname: Chen
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Gao
authors.forename: Wenbing
authors.initials: W
authors.lastname: Yao
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011817:Rabbits; D044967:Serum / Q000276:immunology; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D014443:Tyrosine / Q000235:genetics; D014503:Urate Oxidase / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D014527:Uric Acid / Q000378:metabolism
chemical_list: C476995:3-(4-acetylphenyl)-2-aminopropanoic acid; D050505:Mutant Proteins; D014527:Uric Acid; D014443:Tyrosine; D010649:Phenylalanine; D014503:Urate Oxidase; D000604:Amino Acyl-tRNA Synthetases