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

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–hum...

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Autores principales: Xie, Guangrong, Yang, Weizhen, Chen, Jing, Li, Miaomiao, Jiang, Nan, Zhao, Baixue, Chen, Si, Wang, Min, Chen, Jianhua
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881585/
https://www.ncbi.nlm.nih.gov/pubmed/27213357
http://dx.doi.org/10.3390/ijms17050764
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author Xie, Guangrong
Yang, Weizhen
Chen, Jing
Li, Miaomiao
Jiang, Nan
Zhao, Baixue
Chen, Si
Wang, Min
Chen, Jianhua
author_facet Xie, Guangrong
Yang, Weizhen
Chen, Jing
Li, Miaomiao
Jiang, Nan
Zhao, Baixue
Chen, Si
Wang, Min
Chen, Jianhua
author_sort Xie, Guangrong
collection PubMed
description 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 H(1-2)P(3)H(4)P(5-6)H(7-8) was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H(1-2)P(3)H(4)P(5-6)H(7-8) were favorable for further increase of its activity. After site-directed mutagenesis, H(1-2)P(3)H(4)P(5-6)H(7-8) (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.
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spelling pubmed-48815852016-05-27 Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis Xie, Guangrong Yang, Weizhen Chen, Jing Li, Miaomiao Jiang, Nan Zhao, Baixue Chen, Si Wang, Min Chen, Jianhua Int J Mol Sci Article 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 H(1-2)P(3)H(4)P(5-6)H(7-8) was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H(1-2)P(3)H(4)P(5-6)H(7-8) were favorable for further increase of its activity. After site-directed mutagenesis, H(1-2)P(3)H(4)P(5-6)H(7-8) (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. MDPI 2016-05-20 /pmc/articles/PMC4881585/ /pubmed/27213357 http://dx.doi.org/10.3390/ijms17050764 Text en © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Xie, Guangrong
Yang, Weizhen
Chen, Jing
Li, Miaomiao
Jiang, Nan
Zhao, Baixue
Chen, Si
Wang, Min
Chen, Jianhua
Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis
title Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis
title_full Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis
title_fullStr Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis
title_full_unstemmed Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis
title_short Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis
title_sort development of therapeutic chimeric uricase by exon replacement/restoration and site-directed mutagenesis
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881585/
https://www.ncbi.nlm.nih.gov/pubmed/27213357
http://dx.doi.org/10.3390/ijms17050764
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