Establishing recombinant production protocols for Snake Venom Metalloproteinases to facilitate development of next-generation snakebite treatment

Abstract

Snakebite envenomation, classified by the WHO as a Neglected Tropical Disease, causes more than 100,000 deaths and 400,000 severe disabilities yearly. Current antivenoms have remained largely unchanged for more than a century and are moderately effective, expensive and prone to severe adverse effects. Snake venom is a complex mixture of toxins, synergistically working to induce necrosis, haemorrhage, paralysis and ultimately death in victims. The most potent toxins are snake venom metalloproteinases (SVMPs), however their research has been severely impeded by the inability to efficiently produce these proteins recombinantly. To overcome this bottleneck, I established a baculovirus/insect cell expression protocol using the MultiBac system to successfully produce SVMPs as a zymogen, using a native N-terminally fused prodomain which blocks the active site through a cysteine switch heptapeptide motif located in the prodomain. Activation of the zymogens using Zn2+ resulted in active SVMPs which I characterised through a range of activity assays. In addition to the prodomain and metalloproteinase domain, SVMPs can contain disintegrin domains which are also found in snake venom. I established recombinant production of disintegrins, SVMP prodomain and prodomain-derived β-barrel proteins in E. coli, enabling future experiments to elucidate SVMP domain structure and function. In our quest to develop a potent inhibitor of SVMPs, I explored whether the cysteine switch comprising propeptide could be repurposed as an SVMP inhibitor when displayed in multiple (60) copies on the surface of the ADDomer, a thermostable, synthetic, human adenovirus-derived virus-like nanoparticle engineered for multiepitope display. Independent from that work, during the COVID-19 pandemic, when only SARS-CoV-2 related research could be carried out, I contributed to engineering ADDomer-based vaccine candidates, displaying multiple copies of a SARS-CoV-2 Spike protein receptor binding domain derived immunogenic epitope. Finally, I developed a low temperature baculovirus/insect cell expression protocol to maximise soluble protein yield of ADDomer-based nanoparticles.

Date of Award	10 Dec 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Imre Berger (Supervisor) & J L R Anderson (Supervisor)