21 research outputs found
Modular assembly of proteins on nanoparticles
Generally, the high diversity of protein properties necessitates the development of unique nanoparticle bio-conjugation methods, optimized for each different protein. Here we describe a universal bio-conjugation approach which makes use of a new recombinant fusion protein combining two distinct domains. The N-terminal part is Glutathione S-Transferase (GST) from Schistosoma japonicum, for which we identify and characterize the remarkable ability to bind gold nanoparticles (GNPs) by forming gold–sulfur bonds (Au–S). The C-terminal part of this multi-domain construct is the SpyCatcher from Streptococcus pyogenes, which provides the ability to capture recombinant proteins encoding a SpyTag. Here we show that SpyCatcher can be immobilized covalently on GNPs through GST without the loss of its full functionality. We then show that GST-SpyCatcher activated particles are able to covalently bind a SpyTag modified protein by simple mixing, through the spontaneous formation of an unusual isopeptide bond
Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher
SpyTag is a short peptide that forms an isopeptide bond upon encountering its protein partner SpyCatcher. This covalent peptide interaction is a simple and powerful tool for bioconjugation and extending what protein architectures are accessible. Here we review the origin and mechanism of SpyTag/SpyCatcher, focusing on recent innovative applications. Ligation of targeting-antibody with antigen provided a simple route to vaccine generation. SpyRings, from head-to-tail cyclisation, gave major enhancements in enzyme resilience. Linking multiple SpyCatchers gave dendrimers for T-cell activation or Spy networks forming hydrogels for stem cell culture. Synthetic biology applications include integrating amyloid biomaterials with living bacteria, for irreversible derivatisation of biofilms with enzymes or nanoparticles. We also discuss further opportunities to apply and enhance SpyTag/SpyCatcher technology
Labeling surface proteins with high specificity: Intrinsic limitations of phosphopantetheinyl transferase systems
Nanoassembly routes stimulate conflicting antibody quantity and quality for transmission-blocking malaria vaccines
Vaccine development efforts have recently focused on enabling strong immune responses to poorly immunogenic antigens, via display on multimerisation scaffolds or virus like particles (VLPs). Typically such studies demonstrate improved antibody titer comparing monomeric and nano-arrayed antigen. There are many such studies and scaffold technologies, but minimal side-by-side evaluation of platforms for both the amount and efficacy of antibodies induced. Here we present direct comparison of three leading platforms displaying the promising malaria transmission-blocking vaccine (TBV) target Pfs25. These platforms encompass the three important routes to antigen-scaffold linkage: genetic fusion, chemical cross-linking and plug-and-display SpyTag/SpyCatcher conjugation. We demonstrate that chemically-conjugated Qβ VLPs elicited the highest quantity of antibodies, while SpyCatcher-AP205-VLPs elicited the highest quality anti-Pfs25 antibodies for transmission blocking upon mosquito feeding. These quantative and qualitative features will guide future nanoassembly optimisation, as well as the development of the new generation of malaria vaccines targeting transmission