Soluble expression of bioactive recombinant porcine-human chimeric uricase mutant employing MBP-SUMO fusion system
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 e...
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Veröffentlicht in: | Protein expression and purification 2022-01, Vol.189, p.105978-105978, Article 105978 |
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Zusammenfassung: | 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.
•The highlight of our research is to introduce a method to improve the insoluble expression into a soluble form of recombinant porcine-human chimeric uricase (dHU-wPU) in E. coli to reduce the difficulty of production.•At the same time, the same method is also applicable to mammalian uricases, such as wild porcine uricase (wPU). |
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ISSN: | 1046-5928 1096-0279 |
DOI: | 10.1016/j.pep.2021.105978 |