Abstract
Proteases are multi-functional proteolytic enzymes that have complex roles in human health and disease. Therefore, the development of protease biosensors can be beneficial to global health applications. To this end, we developed Advanced proteoLytic detector PolyHydroxyAlkanoates (AL-PHA) beads – a library of over 20 low-cost, biodegradable, bioplastic-based protease biosensors. Broadly, these biosensors utilise PhaC-reporter fusion proteins that are bound to microbially manufactured polyhydroxyalkanoate beads. In the presence of a specific protease, superfolder green fluorescent reporter proteins are cleaved from the AL-PHA beads – resulting in a loss of bead fluorescence. The Tobacco Etch Virus (TEV) AL-PHA biosensor detected the proteolytic activity of at least 1.85 pM of AcTEV. AL-PHA beads were also engineered to detect cercarial elastase from Schistosoma mansoni-derived cercarial transformation fluid (SmCTF) samples, as well as cancer-associated metalloproteinases in extracellular vesicle and cell-conditioned media samples. We envision that AL-PHA beads could be further developed for use in resource-limited settings.
Highlights
Synthetic biology is an established scientific field based upon engineering design principles, that has led to innovations in the development of biosensors and bioreporters geared towards global health applications [1,2]
In the case of schistosomiasis, a neglected tropical disease that affects over 250 million people worldwide [13,14,15], the invasive Schistosoma cercariae release a cocktail of proteases, including elastase, that help the parasite invade their host through the skin [16,17]
Recent synthetic biology approaches have led to the development of more sophisticated modelling-led design strategies, the embedding of protease biosensors within smart materials and increasingly complex whole-cell bioreporters [4,5,19,22,23]
Summary
Synthetic biology is an established scientific field based upon engineering design principles, that has led to innovations in the development of biosensors and bioreporters geared towards global health applications [1,2]. Exemplars of proteases that have evolved to serve complex biological functions can be found within the matrix metalloproteinase (MMP), the A Disintegrin And Metalloproteinase (ADAM) and A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) protease families [7]. Members of these protease families contribute to an array of biological processes including: cellular metabolism, cell-signalling, cell-migration, immunomodulation and tissue remodelling [8,9]. In the case of schistosomiasis ( known as bilharzia or snail fever), a neglected tropical disease that affects over 250 million people worldwide [13,14,15], the invasive Schistosoma cercariae release a cocktail of proteases, including elastase, that help the parasite invade their host through the skin [16,17]
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