Objective A prokaryotic expression system of α-Hemolysin (HLα), one of key virulence factors of Staphylococcus aureus, was constructed to prepare purified recombinant HLα protein (rHLα) with natural biological activity for drug development against the infection.

Methods The recombinant plasmid pET30a-hlα was transformed into Escherichia coli BL21(DE3). After expression induction, the inclusion bodies were denatured, purified, and refolded. Hemolytic activity, ability to cause cell membrane damage, and activating effect on host membrane receptor, a disintegrin and metalloprotease 10 (ADAM10), of the recombinant protein were evaluated by hemolysis assays, lactate dehydrogenase (LDH) release assays, and western blotting.

Results The pET30a-hlα recombinant expression system was successfully constructed, and the target protein with a molecular weight of approximately 43 kDa obtained. The rHLα was verified to possess significant hemolytic activity, be able to effectively damage cell membranes at concentrations ≥6.3 μg·mL⁻¹, and be capable of inhibiting cellular viability at concentrations ≥50 μg·mL⁻¹. In addition, it significantly activated the ADAM10 expression with the toxic effect intensified at increased concentrations.

Conclusion An efficient and stable rHLα expression and purification system was successfully established that made the recombinant protein with intact biological activity available for further study on the pathogenic mechanism of HLα and development of effective drugs against the bovine mastitis infection.