کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8330199 | 1540238 | 2016 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
DNA shuffling of uricase gene leads to a more “human like” chimeric uricase with increased uricolytic activity
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
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![عکس صفحه اول مقاله: DNA shuffling of uricase gene leads to a more “human like” chimeric uricase with increased uricolytic activity DNA shuffling of uricase gene leads to a more “human like” chimeric uricase with increased uricolytic activity](/preview/png/8330199.png)
چکیده انگلیسی
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 ± 0.04 μM and 2.67 sâ1 μMâ1 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: International Journal of Biological Macromolecules - Volume 82, January 2016, Pages 522-529
Journal: International Journal of Biological Macromolecules - Volume 82, January 2016, Pages 522-529
نویسندگان
Jing Chen, Nan Jiang, Tao Wang, Guangrong Xie, Zhilai Zhang, Hui Li, Jing Yuan, Zengxian Sun, Jianhua Chen,