Efficient production of a novel recombinant fusion protein of EIEC effector IpaD and EGFP: Biophysical characterization and functional studies

Abstract

The conserved invasion plasmid antigen D (IpaD) protein demonstrates broad protective capabilities against bacillary dysentery caused by Enteroinvasive Escherichia coli (EIEC) and Shigella. However, the instability of the IpaD protein at room temperature limits its therapeutic potential. The stabilization and efficient production of functional recombinant proteins remain critical challenges in therapeutic and vaccine development. This study presents a novel fluorescence fusion strategy for producing a stable IpaD-EGFP recombinant protein using a flexible linker (GGGGS)₃. The fusion technique enhances the expression level (∼53 %), solubility (∼77 %), and stability of the IpaD-EGFP fusion protein. Biophysical characterization studies suggest that the IpaD-EGFP fusion protein is stable at refrigerated temperatures for extended periods and up to 1 month at 25 °C. The IpaD-EGFP protein triggers apoptosis in Raw 267.4 cells through activation of caspases 3/7. The protein also induces antibody response in BALB/c mice indicating its immunogenicity. Together, these findings indicate that IpaD-EGFP generated in this study is a potential approach for the design and production of stable IpaD-based protein therapeutics, breaking the expensive “cold chain” of continuous refrigeration. Fusion approach significantly enhanced the solubility, yield, and stability of IpaD, while enabling efficient purification.