Application of a Novel Fusion Tag System for Enhanced Soluble Expression of Recombinant Proteins in Escherichia coli

Background The Escherichia coli expression system is widely used for recombinant protein production, but its utility is often limited by the formation of insoluble inclusion bodies. Although fusion tags can enhance solubility, their effectiveness varies unpredictably across different target proteins, and the optimal tag must typically be determined empirically. Results Here, we developed a novel fusion tag system for the high-throughput screening of soluble protein expression in E. coli . This system consists of eight medium-sized, TEV-cleavable fusion tags (ArsC, Crr, DsbA, Ecotin, MsyB, SlyD, Snut, and YjgD) cloned into a standardized pET-28b(+) backbone. We systematically evaluated the impact of these tags on the solubility and function of three model proteins (eGFP, EcFabG, and Mals) and six challenging proteins (PulA, NodE, FabF1XL, FabF2XL, FabZXL, and FabGXL). Our results demonstrated that the efficacy of each tag was highly protein-dependent. Notably, tags such as MsyB and Snut dramatically increased the soluble proportion of eGFP from 15% to over 85%, while the SlyD tag significantly enhanced both the solubility and activity of Mals. For several difficult-to-express proteins, soluble expression was only achieved with specific tags, highlighting the critical importance of tag selection. Conclusions Our study presents a versatile and efficient platform for the rapid production of soluble recombinant proteins. By enabling parallel screening of multiple fusion partners, this system facilitates the identification of optimal conditions for enhancing protein solubility and function, thereby addressing a key bottleneck in recombinant protein applications.