Purification of an inactive variant alpha-toxin HlaH35LH48L expressed in Escherichia coli and generation of antiserum in mice

Abstract

Alpha-toxin (Hla) is a major cytotoxic agent produced by Staphylococcus aureus, which plays a significant role in the pathogenesis of various infections and is regarded as a promising antigen for vaccine development. This study aims to express Hla_H35LH48L fused with His-tag in Escherichia coli BL21(DE3), purification of the inactive form, and generation of antiserum in mice. The survey results indicated that the appropriate conditions for expressing the
Hla_H35LH48L protein were at 30°C with an IPTG concentration of 0.05 mM, and harvesting 8 hours post-induction. Based on affinity chromatography between the 6xHis tag and Ni-NTA, the Hla_H35LH48L protein was purified, obtaining a concentration of 1.17 mg/ml with a purity of 96%. Activity assays for the purified protein revealed that the Hla_H35LH48L variant lost its ability to lyse rabbit red blood cells. After four injections in mice with a dose of 0.05 mg of Hla_H35LH48L protein per mouse, the resulting antiserum was able to recognise the antigen with an antibody titer of 1:320,000. The results of this study serve as an important premise for further studies to neutralise Hla in vivo and develop a vaccine against S. aureus.