title: Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis.
abstract: The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more "human-like" uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine-human uricase with higher homology to deduced human uricase (dHU) and increased uricolytic activity. In an exon replacement study, substitution of exon 6 in wild porcine uricase (wPU) gene with corresponding exon in dhu totally abolished its activity. Substitutions of exon 5, 3, and 1-2 led to 85%, 60%, and 45% loss of activity, respectively. However, replacement of exon 4 and 7-8 did not significantly change the enzyme activity. When exon 5, 6, and 3 in dhu were replaced by their counterparts in wpu, the resulting chimera H1-2P₃H₄P5-6H7-8 was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H1-2P₃H₄P5-6H7-8 were favorable for further increase of its activity. After site-directed mutagenesis, H1-2P₃H₄P5-6H7-8 (E24D &amp; E83G) with increased homology (91.45%) with dHU and higher activity and catalytic efficiency than the FDA-approved porcine-baboon chimera (PBC) was obtained. It showed optimum activity at pH 8.5 and 35 °C and was stable in a pH range of 6.5-11.0 and temperature range of 20-40 °C.
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, No. 24, Tong Jia Xiang, Nanjing 210009, China. 15311030132@stu.cpu.edu.cn.
authors.forename: Guangrong
authors.initials: G
authors.lastname: Xie
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, No. 24, Tong Jia Xiang, Nanjing 210009, China. 14211030582@stu.cpu.edu.cn.
authors.forename: Weizhen
authors.initials: W
authors.lastname: Yang
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, No. 24, Tong Jia Xiang, Nanjing 210009, China. 1020071789@cpu.edu.cn.
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, No. 24, Tong Jia Xiang, Nanjing 210009, China. 15211030519@stu.cpu.edu.cn.
authors.forename: Miaomiao
authors.initials: M
authors.lastname: Li
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, No. 24, Tong Jia Xiang, Nanjing 210009, China. 13211030516@stu.cpu.edu.cn.
authors.forename: Nan
authors.initials: N
authors.lastname: Jiang
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, No. 24, Tong Jia Xiang, Nanjing 210009, China. 1244724@stu.cpu.edu.cn.
authors.forename: Baixue
authors.initials: B
authors.lastname: Zhao
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, No. 24, Tong Jia Xiang, Nanjing 210009, China. 15211030583@stu.cpu.edu.cn.
authors.forename: Si
authors.initials: S
authors.lastname: Chen
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, No. 24, Tong Jia Xiang, Nanjing 210009, China. minwang@cpu.edu.cn.
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, No. 24, Tong Jia Xiang, Nanjing 210009, China. jhchen@cpu.edu.cn.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Chen
mesh_terms: D000818:Animals; D005091:Exons; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013552:Swine; D014503:Urate Oxidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D014503:Urate Oxidase
keywords: exon replacement/restoration; site-directed mutagenesis; uricase