Biology subjects

Harding, M. D.

Publications and source records attributed to Harding, M. D..

4 recordsLinked to original sources

bioRxiv2026

Selective and Efficient Functionalization of P22 Virus-Like Particles Using an Asparaginyl Ligase

Protein cages provide useful scaffolds for nanoscale engineering due to their highly ordered structures and in vivo self-assembly. These scaffolds are amendable to late-stage conjugation, enabling expansion in functionality. However, many conjugation techniques either lack site-selectivity, require unnatural amino acid incorporation, or have bulky recognition motifs to facilitate ligation reactions. Here, an asparaginyl endopeptidase (AEP) enzyme with ligase activity is employed for the highly efficient functionalization of virus-like particles (VLPs) from Salmonella Typhimurium bacteriophage P22. The capacity of this enzyme to conjugate peptides and proteins onto assembled P22 VLPs under mild reaction conditions, via a minimal extension to the P22 coat protein C-terminus, is demonstrated. We extend the reaction efficiency to facilitate a one-pot dual-functionalization reaction whereby two therapeutically relevant receptor targeting domains are conjugated to P22 VLPs in a single step. Finally, we demonstrate the potential for AEP-mediated bioconjugation to bestow P22 VLPs with receptor-binding functionality in vitro. This work demonstrates the efficacy of AEP ligases as bioconjugation tools for site-selective functionalization of large molecular assemblies like VLPs.

biochemistry↗

bioRxiv2026

Bacteriophage P22 virus-like particles as nanoscale protein scaffolds for plant synthetic biology

Advancing the utility of plant synthetic biology requires the continued development of protein engineering tools. Self-assembling protein compartments, such as virus-like particles (VLPs), provide versatile scaffolds for synthetic biology. However, few plant-expressed VLPs have demonstrated broad amenability to protein engineering, restricting their applications to specific contexts. Here, the Salmonella typhimurium bacteriophage P22 VLP is explored as a novel protein scaffold for plant synthetic biology, demonstrating its application in a eukaryote for the first time. Through transient expression in the biofactory plant Nicotiana benthamiana, the capacity for P22 VLPs to correctly assemble and selectively encapsulate recombinant protein cargo is demonstrated. The durability of this protein scaffold is explored, through co-encapsulation of multiple cargo protein species and by encapsulation through direct fusion to the P22 coat protein. Finally, the ability to simultaneously program cargo encapsulation and external protein display on P22 VLPs in vivo is demonstrated through SpyTag/SpyCatcher-mediated protein conjugation. This work demonstrates the broad utility of P22 VLPs as nanoscale protein scaffolds for plant synthetic biology.

synthetic biology↗

bioRxiv2024

A protein cage system for producing bioactive peptides in Escherichia coli

New therapeutics are urgently needed to curb the spread of drug-resistant diseases. Bioactive peptides (BAPs), including antimicrobial peptides, are emerging as an exciting new class of compounds with advantages over current drug modalities, especially small molecule drugs that are prone to resistance development. Here we evaluated a bacteriophage P22 virus-like particle (VLP) system where BAPs are encapsulated as fusion proteins with the P22 scaffold protein (SP) within self-assembling protein cages in Escherichia coli. Representative peptides from three structurally distinct classes of BAPs were successfully encapsulated into P22 VLPs at high cargo to VLP coat protein (CP) ratios that corresponded to interactions between the compact electropositive structures of the SP-BAPs and electronegative regions on the inward facing surface of CP subunits. However, high loading densities did not correspond to improved SP-BAP yields. An unexpected finding of this study was that while encapsulation alleviated negative effects of SP-BAPs on E. coli growth, the P22 scaffold protein acted as a sufficient fusion partner for accumulating BAPs, and co-expression of the CP did not further improve SP-BAP yields. Nevertheless, encapsulation in VLPs provided a useful first step in the purification pipeline for producing both linear and cyclic recombinant (r)BAPs that were functionally equivalent to their synthetic counterparts. Further efforts to optimise expression ratios of CP to SP-BAP fusions will be required to realise the full potential of encapsulation for protecting expression hosts and maximizing rBAP yields.

synthetic biology↗

bioRxiv2021

Rational domestication of a plant-based recombinant expression system expands its biosynthetic range.

Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead and an orally active analgesic are demonstrated.

synthetic biology↗