“The production of eight plasmid expression vectors through Gibson assembly and the optimization of the enzymatic cleavage reaction between tobacco etch virus (TEV) and the model fusion protein, Nus-G”
R. Marshall Werner
Snake venom metalloproteinases (SVMPs), Phospholipase 2As (PLA2s)
Kennard, Elizabeth, "“The production of eight plasmid expression vectors through Gibson assembly and the optimization of the enzymatic cleavage reaction between tobacco etch virus (TEV) and the model fusion protein, Nus-G”" (2021). Student Summer Scholars Manuscripts. 219.
Snake envenomization is a neglected public health issue that effects large portions of the world, as venomous snakes are found on six continents (World Health Organization,2007). With 2-3 million people bitten globally every year leading to an estimated 125,000 deaths and 400,000 amputations, researching effective treatments is important. Several Mammalian species have a natural resistance to snake venom (Voss, R.S et al.,2012; Bastos, V.A. et al.,2016). The North American Opossum (D. virginiana) produces proteins that inhibit certain snake venom components including snake venom metalloproteinases (SVMPs) and phospholipase 2As (PLA2s) (Catanese, J.J. et al.,1992; Perales, J. et al.,1994; Farah, M.D.F.L. et al.,1996; Neves-Ferreira, A.G. et al.,2000; Jurgilas, P.I.B. et al.,2003). The goal of studying the protein inhibitors produced by the North American Opossum is to better understand the mechanisms that cause inhibition thus leading to the development of effective inhibitors. Eight plasmid expression vectors containing 6-histidine tags and maltose binding proteins (MBP), the coding sequence for either DM43 (a SVMPI) or DM64 (aPLA2 inhibitor), and a Tobacco etch virus (TEV) cleavage site were constructed through Gibson Assembly. Four constructs were used to produced either MBP-DM43 or MBP-DM64 in E. coli. The optimization of the enzymatic cleavage reaction between TEV and a model fusion protein with a TEV cleavage sites, MBP-NusG, was accomplished using 2 experiments. These experiments optimized the duration, temperature, and amount of TEV used to conduct the cleavage reaction. For our purposes, the optimal conditions include the shortest duration and lowest temperature in which the reaction proceeds to completion. Reactions conducted at 4°C with 5.5ug TEV for 1hr gave optimal cleavage of MBP-NusG providing useful conditions for future reactions.