Purification of active staphylococcal alpha-toxin produced in <i>Bacillus subtilis</i>

The Gram-positive bacterium Staphylococcus aureus, a multidrug-resistant pathogen, poses a significant threat to human and animal health. Its virulence is largely attributed to the production of alpha-toxin (Hla), a potent cytotoxic protein that forms pores in cell membranes, leading to lysis. This is the first study to report the successful expression and purification of wild-type Hla (HlaWT) using Bacillus subtilis-a safe, genetically stable, and endotoxin-free host system. A vector was constructed harboring the hla gene fused to Strep-tag II at the C-terminus under the control of a strong isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible Pgrac212 promoter in a pHT expression vector for B. subtilis. Following IPTG induction, extracellular Hla was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Western blotting using anti-Hla polyclonal antibodies. The Hla protein was purified in a single step using a Strep-Tactin column and showed a high response to the antibodies, as confirmed by Enzyme-Linked Immunosorbent Assay (ELISA). Hemolytic measurements using rabbit red blood cells demonstrated that Hla exhibits potent hemolytic activity. These findings demonstrate the potential of B. subtilis as an effective host for producing biologically active extracellular toxins and provide a scalable, safe platform for HlaWT production in pathogenesis research, diagnostics, immunology, drug screening, and toxoid vaccine development.