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AL-PHA beads: bioplastic-based protease biosensors for global health applications

View ORCID ProfileRichard J. R. Kelwick, Alexander J. Webb, Yizhou Wang, Amelie Heliot, Fiona Allan, Aidan M. Emery, Michael R. Templeton, Paul S. Freemont
doi: https://doi.org/10.1101/2020.06.18.159921
Richard J. R. Kelwick
1Section of Structural and Synthetic Biology, Department of Infectious Disease, Imperial College London, London, SW7 2AZ, UK
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  • ORCID record for Richard J. R. Kelwick
Alexander J. Webb
1Section of Structural and Synthetic Biology, Department of Infectious Disease, Imperial College London, London, SW7 2AZ, UK
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Yizhou Wang
1Section of Structural and Synthetic Biology, Department of Infectious Disease, Imperial College London, London, SW7 2AZ, UK
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Amelie Heliot
1Section of Structural and Synthetic Biology, Department of Infectious Disease, Imperial College London, London, SW7 2AZ, UK
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Fiona Allan
2Department of Life Sciences, Natural History Museum, London, SW7 5BD, UK
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Aidan M. Emery
2Department of Life Sciences, Natural History Museum, London, SW7 5BD, UK
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Michael R. Templeton
3Department of Civil and Environmental Engineering, Imperial College London, SW7 2AZ, UK
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Paul S. Freemont
1Section of Structural and Synthetic Biology, Department of Infectious Disease, Imperial College London, London, SW7 2AZ, UK
4The London Biofoundry, Imperial College Translation & Innovation Hub, White City Campus, 80 Wood Lane, London W12 0BZ, UK
5UK Dementia Research Institute Care Research and Technology Centre, Imperial College London, Hammersmith Campus, Du Cane Road, London, W12 0NN
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  • For correspondence: p.freemont@imperial.ac.uk
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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.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted June 18, 2020.
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AL-PHA beads: bioplastic-based protease biosensors for global health applications
Richard J. R. Kelwick, Alexander J. Webb, Yizhou Wang, Amelie Heliot, Fiona Allan, Aidan M. Emery, Michael R. Templeton, Paul S. Freemont
bioRxiv 2020.06.18.159921; doi: https://doi.org/10.1101/2020.06.18.159921
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AL-PHA beads: bioplastic-based protease biosensors for global health applications
Richard J. R. Kelwick, Alexander J. Webb, Yizhou Wang, Amelie Heliot, Fiona Allan, Aidan M. Emery, Michael R. Templeton, Paul S. Freemont
bioRxiv 2020.06.18.159921; doi: https://doi.org/10.1101/2020.06.18.159921

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