Cloning and Expression of Therapeutic Enzyme, Aspergillus Flavus Uricase in E. coli
Publish Year: 1395
نوع سند: مقاله ژورنالی
زبان: English
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JR_ZUMS-24-106_011
تاریخ نمایه سازی: 11 اردیبهشت 1400
Abstract:
Background and Objective: Uricase (EC ۱.۷.۳.۳) is an enzyme which catalyses uric acid to H۲O۲ and alantoin and is utilized in the treatment of hyperuricemia or gout. The aim of this study was cloning of Aspergillus flavus uricase synthetic gene into pET-۲۸(a) and its recombinant expression in E. coli
Materials and Methods: The coding sequence of uricase retrieved from gene bank and once optimized, the gene synthesis was ordered. Then, the synthetic gene subcloned between NcoI and XhoI within the vector. The proper subcloning was confirmed by enzymatic digestion check, PCR and finally DNA sequencing. The construct, then chemically transformed into E. coli BL۲۱ and induced by IPTG which followed by overexpression optimization. For enzyme expression evaluation, protein gel electrophoresis was utilized.
Results: Restriction check, PCR and sequencing indicated that the coding sequence of enzyme was cloned in the desired point of vector. The optimized condition obtained for enzyme expression was IPTG ۱mM for ۶ h at ۲۲ °C. Enzyme molecular weight was determined ۳۴.۵ kDa by electrophoresis. Evaluation of uricase specific activity showed that the expressed enzyme is ۷.۶۹ U/mg and it is catalytically active.
Conclusion: The results of the current research showed that the uricase gene could be simply cloned and expressed in pET system. Enzyme specific activity determination exhibited that the enzyme was produced as a soluble protein and catalytically active in E. coli cytosole.
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Authors
مهدی ایمانی
Dept. of Biochemistry, Basic Sciences Group, Faculty of Veterinary, Urmia University, Urmia, Iran
محمد پاژنگ
Dept. of Cellular and Molecular Biology, Faculty of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran
سمیه میرزائینیا
Dept. of Cellular and Molecular Biology, Faculty of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran
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