Cloning, Expression, and Purification of Recombinant Urate Oxidase Using Intein Sequence and Elastin-like Protein | ||
| Pathobiology Reserach | ||
| Article 2, Volume 21, Issue 1, 2018, Pages 7-13 PDF (484.91 K) | ||
| Document Type: Original Research | ||
| Authors | ||
| M. Azizi; S. Enayati* ; M. Salmasizadeh; نجمه زارعی; V. Khalaj | ||
| Medical Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran | ||
| Abstract | ||
| Aims: Expression and purification of recombinant Urate oxidase as well as other pharmaceutical proteins are a costly process. The use of non-chromatographic methods in the purification process can reduce production costs. So, the aim of this study was cloning, expression, and purification of recombinant urate oxidase, using intein sequence and elastin-like protein (ELP). Materials and Methods: In the present experimental research, the synthetic urate oxidase gene was cloned in downstream of ELP–Intein in an expression plasmid. After cultivating bacteria containing urate oxidase expression plasmid, the soluble fraction of cell lysate was separated from insoluble portion, using centrifugation. The recombinant urate oxidase was precipitated by adding salt and incubation at 37°C. The resulting precipitant was resuspended in cleavage buffer and the urate oxidase was separated from ELP through Intein moiety. Adding salt for the second time and incubation at 37°C resulted in separation of urate oxidase from fusion partner. After expressing and purifying the urate oxidase, purity and biological activity of recombinant urate oxidase were compared to standard drug. Findings: The recombinant urate oxidase was expressed and purified using ELP and Intein tags as fusions partners. The rescombinant enzyme showed a purity of %90 equal to 1.89 mg.ml-1 based on protein concentration and 1.64 mg.ml-1 based on activity. Conclusion: Using Intein sequence and Elastin-like protein, together with the Urate Oxidase enzyme helps to express and purify the enzyme without need for a chromatographic method. | ||
| Keywords | ||
| Urate Oxidase; Purification; Intein; Elastin | ||
| References | ||
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