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Int. J. Mol. Sci. 2016, 17(5), 764; doi:10.3390/ijms17050764

Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis

School of Life Science and Technology, China Pharmaceutical University, No. 24, Tong Jia Xiang, Nanjing 210009, China
These authors contributed equally to this paper.
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Academic Editor: Terrence Piva
Received: 30 March 2016 / Revised: 1 May 2016 / Accepted: 6 May 2016 / Published: 20 May 2016
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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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 & 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. View Full-Text
Keywords: uricase; exon replacement/restoration; site-directed mutagenesis uricase; exon replacement/restoration; site-directed mutagenesis
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MDPI and ACS Style

Xie, G.; Yang, W.; Chen, J.; Li, M.; Jiang, N.; Zhao, B.; Chen, S.; Wang, M.; Chen, J. Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis. Int. J. Mol. Sci. 2016, 17, 764.

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