Efficient production of functional proaerolysin in E. coli

Proaerolysin is a bacterial toxin produced by Aeromonas hydrophila that specifically binds to GPI-anchored proteins on the plasma membrane, forming transmembrane pores that induce cell death. Leveraging this unique property, proaerolysin is widely used in diagnostic tests for paroxysmal nocturnal hemoglobinuria (PNH), a disease caused by somatic mutations in the PIGA, a gene involved in the biosynthesis of GPI anchors. Beyond diagnostics, proaerolysin has recently been applied in basic research, including its use as a counter-selection agent in genetic manipulations and as an engineered nanopore for single-molecule detection. Although the bacterial expression and purification of proaerolysin have been previously reported, the yields were low due to its low solubility. Here, we demonstrate that using the SHuffle E. coli strain, which facilitates the disulfide bond formation in the cytoplasm, significantly improves the solubility and proper folding of proaerolysin. We achieved a high yield of proaerolysin, approximately 3 mg from a 50 mL bacterial culture, with a purity exceeding 99 %. The functionality of recombinant proaerolysin was confirmed through testing in mouse embryonic stem cells (mESCs), demonstrating that this high-yield production method provides a reliable and cost-effective source of functional proaerolysin for a wide range of biotechnological applications.