Abstract

This study establishes an in situ refolding–based high-throughput screening strategy that enables directed evolution of eukaryotic enzymes expressed as inactive inclusion bodies in Escherichia coli. Guided by bioinformatic analysis, the catalytic domain of human DNase I was selected for mutagenesis, generating a library of 1,051 variants. A streamlined workflow—comprising microplate-based cultivation, induction, cell lysis, in situ denaturation with alkaline buffer containing β-mercaptoethanol, refolding with arginine, and activity detection—allowed efficient screening directly from insoluble expression. From this library, a DNase I mutant carrying N78T and V90N substitutions exhibited a 4.1-fold increase in enzymatic activity compared with the wild type. The same strategy applied to benzonase yielded a mutant with a 40% activity improvement, demonstrating the method’s generality. Collectively, these results show that in situ refolding enables rapid identification of functional mutants from eukaryotic proteins produced as inclusion bodies, thereby improving the efficiency of directed evolution for otherwise challenging enzyme targets.