ABSTRACT
Proteases are multi-functional, proteolytic enzymes that have complex roles in human health and disease. Detecting the activities of proteases can lead to important insights into communicable and non-communicable diseases. Therefore, the development of protease detection strategies can be beneficial to an array of global health applications. To this end, we developed Advanced proteoLytic detector PolyHydroxyAlkanoates (AL-PHA) beads – a library of low-cost, biodegradable, bioplastic-based protease biosensors. Broadly, these biosensors utilise PhaC-reporter fusion proteins that are bound to microbially manufactured polyhydroxyalkanoate (PHA) bioplastic beads. These PhaC-fusions also incorporate modular specific protease cleavage sites. In the presence of a specific protease, superfolder green fluorescent (sfGFP) reporter proteins are cleaved off of the AL-PHA beads - resulting in a loss of bead fluorescence. These AL-PHA biosensors were initially optimised using a commercially available Tobacco Etch Virus (TEV) protease. Our third generation TEV biosensor (PhaC-112L-T-G) detected 0.5 U (1.85 pM) of AcTEV activity and 10 units of AcTEV protease activity resulted in a visually noticeable loss in AL-PHA bead fluorescence. AL-PHA beads also detected 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 adapted towards a low-cost and high-throughput protease detection assay for global health applications.
Competing Interest Statement
The authors have declared no competing interest.
