title: DNA shuffling of uricase gene leads to a more "human like" chimeric uricase with increased uricolytic activity.
abstract: Urate oxidase (Uox) is the enzyme involved in purine metabolism. Pseudogenization of Uox gene is the underlying mechanism of hyperuricemia and gout in human. Although Uox from various microorganisms has been used in clinical practice for many years, its application is limited by potential immunogenicity. In order to develop a more "human like" uricase, DNA shuffling was used to create chimeric uricase with both improved enzymatic activity and increased homology with deduced human uricase (dHU) gene. By using wild porcine uricase (wPU) gene and dhu as parental genes, a diverse chimeric library was generated. After preliminary screening by a "homebrew" high throughput protocol, approximately 100 chimeras with relatively high enzymatic activity were obtained. By further activity comparison of the purified enzymes, chimera-62 with increase in both activity and homology with dHU compared with wPU was selected. Its Km and catalytic efficiency were determined as 9.43±0.04μM and 2.67s(-1)μM(-1) respectively. There were 33 amino acid substitutions in chimera-62 when compared with dHU and 5 substitutions when compared with wPU. By homology modeling and 3-D structure analysis, it was speculated that mutations G248S and L266F contributed to the increased activity of chimera-62 by increasing the stability of α-helix and surface polarity respectively.
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Jiang
authors.affiliation: Department of Neurosurgery, Shanghai 5th People's Hospital, Shanghai Medical College, Fudan University, Shanghai 200240, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Guangrong
authors.initials: G
authors.lastname: Xie
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Zhilai
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.affiliation: School of life science, Faculty of Health and Life science, University of Liverpool, Liverpool, L69 3BX, UK.
authors.forename: Jing
authors.initials: J
authors.lastname: Yuan
authors.affiliation: Department of Pharmacy, The First People's Hospital of Lianyungang, Lianyungang 222002, China. Electronic address: sunzx715@163.com.
authors.forename: Zengxian
authors.initials: Z
authors.lastname: Sun
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China. Electronic address: chenjhnj@163.com.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D032621:DNA Shuffling; D004789:Enzyme Activation; D015870:Gene Expression; D015723:Gene Library; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014503:Urate Oxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014527:Uric Acid / Q000378:metabolism
chemical_list: D014527:Uric Acid; D014503:Urate Oxidase
keywords: DNA shuffling; High-throughput screening; Urate oxidase