Biology subjects

Webb, A. J.

Publications and source records attributed to Webb, A. J..

3 recordsLinked to original sources

bioRxiv2025

Hollow-fibre biomanufacturing and cell-free engineering of HEK293 extracellular vesicles

Extracellular vesicles (EVs) are lipid-delineated nanoparticles that are produced by most cell types. EVs contain complex molecular cargoes that can have useful therapeutic or vaccine immunological effects. Cell-free gene expression systems can be used to produce membrane proteins in vitro, that can co-localise and integrate with exogenously added EVs. To advance this type of cell-free EV engineering we manufactured, isolated and characterised HEK293 cell EVs. These EVs were successfully cell-free engineered with several CD63-based membrane fusion proteins. In our most optimal conditions, up to 4.83 x1011 /ml of HEK293 EVs were successfully cell-free engineered with a fusion membrane protein incorporating CD63 I-shaped membrane-insertion topology transmembrane helix 3 (CD63ITM3) and monomeric green lantern (mGL). Finally, we also demonstrated that nano flow cytometry is a powerful tool for assessing cell-free EV engineering efficiency. In the future cell-free EV engineering could help accelerate future EV discoveries and the development of EV translational applications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/677537v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@2f6b33org.highwire.dtl.DTLVardef@1270337org.highwire.dtl.DTLVardef@442c2aorg.highwire.dtl.DTLVardef@1afabcc_HPS_FORMAT_FIGEXP M_FIG C_FIG Technology readinessCell-free systems are a well-established part of the engineering biology toolkit. Indeed, several applications are already at TRL9 stage, including commercially available cell-free protein synthesis kits, drug screening assays, field-tested medical biosensors, as well as therapeutic antibody manufacturing that has now reached cell-free reaction volume scales of up to 4500 L. However, cell-free extracellular vesicle (EV) engineering is currently at TRL3 / 4 stage. Whilst our study helps to further advance cell-free EV engineering, key challenges remain. To accelerate technology readiness, improvements in scalable EV isolation methods, increased eukaryotic cell-free protein synthesis yields, and assay automation will likely speed up cell-free EV engineering workflows. Furthermore, artificial intelligence-guided design workflows could be used to rapidly create community accessible libraries of EV membrane protein scaffolds, therapeutic, cell targeting and other modular elements for use in EV engineering studies. Finally, the development of EV potency assays for functional assessment of cell-free engineered EVs are needed to progress prototyped designs towards clinical translation. If these challenges are addressed, we envision that cell-free EV engineering will become an important approach in EV biological research and clinical applications. HighlightsO_LICell-free extracellular vesicle (EV) engineering could be utilised to rapidly prototype and test novel biotechnological, vaccine, or therapeutic EVs for foundational or translational applications. C_LIO_LIOur data highlight some of the impacts that hollow fibre-based cell culture, EV isolation methods and different cell-free engineering approaches have on cell-free EV engineering workflows. C_LIO_LIWe demonstrate several characterisation assays and technologies, including nanoflow cytometry, that can be used to assess cell-free EV engineering efficiency. C_LI

synthetic biology↗

bioRxiv2020

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

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.

synthetic biology↗

bioRxiv2017

Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics

The polyhydroxyalkanoates are a group of microbially-produced biopolymers that have been proposed as sustainable alternatives to several oil-derived plastics. However, polyhydroxyalkanoates are currently more expensive to produce than oil-derived plastics and therefore, more efficient production processes would be desirable. Cell-free transcription-translation-based metabolic engineering strategies have been previously used to optimise several different biosynthetic pathways but not the polyhydroxyalkanoates biosynthetic pathways. Here we have developed several Escherichia coli cell-free transcription-translation-based systems for in vitro prototyping of polyhydroxyalkanoates biosynthetic operons, and also for screening relevant metabolite recycling enzymes. These cell-free transcription-translation reactions were customised through the addition of whey permeate, an industrial waste that has been previously used as a low-cost feedstock for optimising in vivo polyhydroxyalkanoates production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by ~20% compared to control reactions that did not include whey permeate. An analysis of pH in our cell-free reactions suggests that the observed increase in GFPmut3b production was likely through enhanced ATP generation, as a consequence of the glycolytic processing of lactose present in whey permeate. We also found that whey permeate enhanced cell-free reactions produced ~3M (R)-3HB-CoA, whilst, coupled cell-free biotransformation/transcription-translation reactions produced a ten-fold greater yield of (R)-3HB-CoA. These reactions were also used to characterise a Clostridium propionicum propionyl CoA transferase enzyme that can recycle Acetyl-CoA. Together our data demonstrate that cell-free approaches can be used to complement in vivo workflows for identifying additional strategies for optimising polyhydroxyalkanoates production.

synthetic biology↗