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bioRxiv · 10.1101/2025.06.27.661748

Efficient Recombinant Production and Functional Characterisation of Cytotoxic and Haemotoxic Snake Venom Metalloproteinases

Hall, S.·Cardoso, I. A.·Wilkinson, M. C.·Carretero, M. M.·Lingappa, S.·Rand, B.·Shen, D.·Boldrini-Franca, J.·Stenner, R.·Balchin, G.·Hus, K. K.·Vincentelli, R.·Mumford, A.·Casewell, N. R.·Berger, I.·Schaffitzel, C.
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Abstract

Snake venoms contain diverse mixtures of toxins that evolved to incapacitate prey, but in humans they cause extensive pathology following snakebite envenomation. In viper venom, some of the most potent toxins are the haemorrhagic and coagulopathic snake venom metalloproteinases (SVMPs). Because venoms contain a SVMP cocktail, and due to their cytotoxicity, SVMP characterizations have been hampered by the lack of purified enzymes. By incorporating their prodomain, which blocks the active SVMP site, we overcame their cytotoxicity and enabled recombinant production of zymogens from all three structurally variable SVMP classes (PI, PII and PIII) using our baculovirus/insect cell expression system. Zymogens were auto-activated by incubation with Zn2+ ions, resulting in prodomain cleavage, PII disintegrin cleavage and PIII prodomain proteolysis. Auto-activated SVMPs were characterized using protein substrate degradation, platelet aggregation and blood coagulation assays, benchmarked to native venom-purified SVMP. Our recombinant zymogen production protocol is generically applicable for the expression of SVMPs, unlocking biomedical use in haematology, and discovery of novel snakebite therapeutics.

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Hall, S., Cardoso, I. A., Wilkinson, M. C., Carretero, M. M., Lingappa, S., Rand, B., Shen, D., Boldrini-Franca, J., Stenner, R., Balchin, G., Hus, K. K., Vincentelli, R., Mumford, A., Casewell, N. R., Berger, I., Schaffitzel, C.. 2025-06-30. Efficient Recombinant Production and Functional Characterisation of Cytotoxic and Haemotoxic Snake Venom Metalloproteinases. https://doi.org/10.1101/2025.06.27.661748

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