An optimized high-pH solubilization strategy for efficient purification and functional recovery of E. coli-expressed...

The heterologous expression of Bacillus thuringiensis (Bt) Cry toxins in Escherichia coli is frequently hindered by the formation of insoluble inclusion bodies, which severely limits subsequent functional and structural characterizations. In this study, we report an optimized in vitro solubilization and purification protocol utilizing a highly alkaline carbonate buffer (pH 11.3) to efficiently recover Cry toxins. Compared to the conventional method, this optimized strategy significantly enhanced the solubilization efficiency, increasing the yields of Cry1Ab and Cry1Fa by approximately 3.6-fold and 2.75-fold, respectively. Furthermore, the method proved highly effective for the structurally divergent, Coleopteran-specific Cry8Ea1 toxin. Following trypsin activation and size-exclusion chromatography, the purified toxins exhibited high purity and successfully retained their native biological functions. Specifically, purified Cry1Ab and Cry1Fa demonstrated potent receptor-dependent cytotoxicity against Sf9 cells heterologously expressing the BmABCC2 receptor, alongside distinct pore-forming activity in artificial liposomes. Utilizing the high-quality purified Cry8Ea1, we further investigated its oligomerization potential in membrane-mimetic environments, capturing specific early-stage assemblies-including detergent-induced "cloverleaf-shaped" trimers and nanodisc-reconstituted "wedge-shaped" dimers-via negative-stain electron microscopy. Overall, this highly efficient and versatile purification paradigm successfully overcomes the technical bottleneck of inefficient inclusion body solubilization during the in vitro preparation of Cry toxins, thereby laying a reliable material foundation for downstream functional applications and high-resolution structural characterization of their pore-forming mechanisms.