Approach
We wanted 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.
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.
The BioBrick 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 BioBrick 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.
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