Soluble expression of bioactive recombinant porcine-human chimeric uricase mutant employing MBP-SUMO fusion system
| Autor: | Xiangwei Yu, Ligang Zhang, Mingjie Tong, Hui Zhao, Qiuling Xie, Zhenlong Zhou, Qiwei Liu, Sheng Xiong |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Urate Oxidase
Swine Recombinant Fusion Proteins Mutant SUMO-1 Protein Gene Expression Hyperuricemia medicine.disease_cause Maltose-Binding Proteins law.invention Bioreactors Affinity chromatography law medicine Escherichia coli Animals Humans Cloning Molecular Chemistry Immunogenicity Urate oxidase Fusion protein In vitro Recombinant Proteins Uric Acid Biochemistry Solubility Mutation Recombinant DNA Biotechnology Plasmids |
| Zdroj: | Protein expression and purification. 189 |
| ISSN: | 1096-0279 |
| Popis: | Urate oxidase is a promising biological medicine for hyperuricemia treatment, but immunogenicity obstructs the development of its clinical application. The recombinant porcine-human chimeric uricase mutant named dHU-wPU is a humanized chimeric uricase based on wild porcine uricase (wPU), which can effectively reduce the limitation of potential immunogenicity with a high homology (92.76%) to deduced human uricase (dHU). Unfortunately, the insoluble expression form of dHU-wPU in E. coli increases the difficulty of production. In this study, we described a more convenient method to efficiently obtain recombinant dHU-wPU protein from E. coli. Combination small ubiquitin-related modifier protein (SUMO) and maltose-binding protein (MBP) was employed to achieve the soluble expression of dHU-wPU. MBP-SUMO-dHU-wPU fusion protein was not only overexpressed in a soluble form, but also showed high purification and cleavage efficiency. Subsequently, we optimized the culture conditions of shake flasks and expanded the production of MBP-SUMO-dHU-wPU fusion protein in a 5 L bioreactor. Finally, about 15 mg of recombinant dHU-wPU was obtained from 1 L M9 fermentation culture by using two-step affinity chromatography, with a SDS-PAGE purity over 90%. In vitro activity analysis showed that dHU-wPU had better ability to catalyze uric acid than wPU. |
| Databáze: | OpenAIRE |
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