Refolding of recombinant Tityus toxins improves antigen quality for their use as immunogens in Antivenom production

Introduction

Approximately 80% of the 8,000 annual envenomation cases reported in Argentina are attributed to scorpion stings, with Tityus carrilloi being the most medically significant species. Antivenom is the only specific treatment for severe cases. It is produced from the plasma of horses hyperimmunized with T. carrilloi venom. However, the venom supply represents a bottleneck in antivenom production. In Tityus serrulatus, a related species, recombinant toxins have been investigated as potential replacements or complements for native venom. A similar approach could be applied to T. carrilloi by identifying key toxin candidates and optimizing expression systems to improve antigenicity. Sodium channel-targeting toxins, which drive the most severe symptoms, have complex structures stabilized by four disulfide bonds. This study evaluated how antigenicity is influenced by refolding conditions that promote native-like conformations.

Methods

Fusion proteins 6xHis_MBP_TsNTxP and 6xHis_MBP_Tt1G were expressed in E. coli Shuffle® cells and purified using immobilized metal affinity chromatography under denaturing conditions. Protein expression, molecular weight, and purity were confirmed via SDS–PAGE and Western blotting. Antigenic recognition was assessed via ELISA using six independent antivenom batches against reduced/alkylated, refolded, and non-refolded protein versions.

Results

Soluble recombinant TsNTxP and Tt1G fused to MBP were successfully expressed in E. coli. All six antivenom batches showed stronger recognition of the refolded proteins, confirming the relevance of conformational epitopes. Moreover, TsNTxP exhibited stronger reactivity than Tt1G, supporting its potential as a complementary immunogen to T. carrilloi venom in antivenom production.

Conclusions

Our findings demonstrate that refolding significantly increased recognition by T. carrilloi antivenom for both TsNTxP (from T. serrulatus) and Tt1G (from T. carrilloi), highlighting the role of conformational epitopes in immune recognition.