Designing a mutant Candida uricase with improved polymerization state and enzymatic activity.

As human uricase has been silenced during evolution, counterparts from other species become an alternative for the treatment of hyperuricemia. Candida uricase is a promising option among them, but its aggregation propensity remains a major obstacle to clinical use. In this study, we designed two mutations according to homology-modeled 3D structure of Candida uricase: Cys249Ser substitution and C-terminal Leu deletion. The wild-type uricase and three mutants containing either or both of the mutations were expressed in Escherichia coli BL21 and validated by mass spectrometry. Size-exclusion chromatography and electrophoresis analysis demonstrated that aggregation was induced by interchain disulfide bonds and could be significantly avoided by Cys249Ser substitution. In combination with Cys249Ser substitution, deletion of Leu increased the enzymatic activity by 8%. Taken together, mutant containing both mutations is chosen as our target protein which is comparatively more suitable for therapeutic use. In addition, homology-modeled 3D structure was proved to be an efficient approach for protein engineering.