Team:DTU-Denmark/Tour Approach
Approach
Venomic studies have enabled the characterization of venom composition, from snakes species of medical importance [1]. From these, it can be shown that the venom between snake species, even within families, is more similar than expected [2]. However, these studies have also shown that there are certain enzymes that are innate to snake families, or much higher in abundance compared to distant species.
Our approach
We applied an alternative approach to detect snake venom circumventing immunoassay based methods. We decided to create a proteolytic enzyme assay based on a linker sequence which contains a cleavage site for proteases that are characteristic for the venom of different snakes.
Our approach to snake venom detection, utilising the proteolytic activity of different venoms, has several potential advantages compared to current methods used for snake venom detection, such as lowers cost, new target substrates and relative quantification of the envenoming.
Choice of snakes
The focus of our project was sub-Saharan Africa, where snakebites are more common than other regions of the continent. As a proof of concept, we attempted to distinguish the snake venoms from the species Bitis gabonica, Bitis arietans and Naja nigricollis.
Our Biosensor
Proteins that give off signals when a linker sequence is cleaved, are not a new invention. FRET (Föster resonance energy transfer) has been used for many years in research, to gain a better understanding of protein-protein interactions.
Our biosensor consists of a fusion protein. Inspired by the design of different FRET reporters, our fusion protein contains two larger protein domains, separated by a flexible linker containing one or more substrate sequences. However, unlike FRET reporters, where fluorescent proteins make up the two larger domains, the two large protein domains in our biosensor are a chromoprotein domain and a binding domain. The linker between the two domains is designed to contain the specific cleavage sites found through screening methods. The binding domain can be used to immobilize the construct, in order to detect cleavage by the release of the chromophore protein.
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References
[1] Fasoli E, Sanz L, Wagstaff S, Harrison RA, Righetti PG, Calvete JJ (2010). Exploring the venom proteome of the African puff adder, Bitis arietans, using a combinatorial peptide ligand library approach at different pHs. J Proteomics.,73(5):932-42.
[2]Calderón-Celis F, Cid-Barrio L, Encinar JR, Sanz-Medel A, Calvete JJ (2017). Absolute venomics: Absolute quantification of intact venom proteins through elemental mass spectrometry. J Proteomics., 164:33-42.
