Purification of snake venom derived phospholipase A2s

Student thesis: Master's ThesisMaster of Science by Research (MScR)

Abstract

Phospholipase A2s (PLA2s) are one of the primary components of viper venoms, accounting for 10-90% of bioactive venom compounds. The large number of different PLA2 isoforms contributes to their multifunctionality and variable physiological impact (such as neurotoxicity, myotoxicity and hemotoxicity), leading to snakebite related mortalities and morbidities.

Snakebite envenoming has been recognised as a Highly Neglected Tropical Disease by the World Health Organisation. Annually, snakebite envenomation affects an estimated 2.7 million people, causing 100,000 fatalities and leaving 400,000 disabled. The ADDovenom project, which this research contributes to, aims to develop an affordable, thermostable, and clinically safe antivenom to target venomous snakebite in Sub-Saharan Africa. Strongly neutralising venom binders will be developed using the ADDomer platform and selective evolution, a process which requires the production of pure, active, snake venom toxins for use as antigens.

In this one-year MScR project, four hemotoxic PLA2s were chosen as toxin targets: PLA2_6, PLA2 _7, PLA2_8 and PLA2_9. These are from the venoms of Echis coloratus (Carpet viper), Echis carinatus (Saw-scaled viper) and Echis ocellatus (Ocellated saw-scaled viper). All four PLA2s were successfully expressed and purified from both bacterial and insect cells, and preliminary activity assays on the four PLA2s were undertaken - high specific activity is important as the purified recombinant PLA2s should match crude snake-venom PLA2s as much as possible. Initial results show that the High Five™ insect cell expression system produces a higher yield of each PLA2, as well as PLA2s with a greater specific activity, than the bacterial BL21(DE3) Escherichia coli expression system.

Future work would involve repeating these experiments to confirm findings, and developing an optimised protocol through which PLA2 production in insect cells could be scaled up. After this, the next step would be using the purified PLA2s as antigens to develop neutralising ADDobody binders via ribosome display.
Date of Award20 Jun 2023
Original languageEnglish
Awarding Institution
  • University of Bristol
SupervisorImre Berger (Supervisor) & Steven G Burston (Supervisor)

Keywords

  • PLA2s
  • ADDovenom
  • snakebite
  • antivenom

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