Efficient production of functional proaerolysin from E.coli

Abstract

Proaerolysin is a bacterial toxin produced by Aeromonas hydrophila that specifically binds to GPI-anchored proteins on the plasma membrane, creating transmembrane pores that lead to cell death within a few hours. 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 gene, which is involved in the biosynthesis of GPI anchors. Additionally, proaerolysin serves as a counter-selection agent in genetic manipulations. Although bacterial expression and purification of proaerolysin have been previously reported, yields were low due to the absence of internal disulfide bonds, which are crucial for protein stability. Here, we demonstrate that using the Shuffle E. coli strain, which facilitates the formation of disulfide bonds 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 of over 99%. The functionality of recombinant proaerolysin was confirmed by testing in mouse embryonic stem cells (mESCs), demonstrating that this high-yield production method offers a reliable and cost-effective source of functional proaerolysin for a wide range of biotechnological applications.