Protein-nanoparticles interaction and assembly
Creators: name: Ma, Wenwei
Publisher: name: University of Lincoln
Year: 2023
Subjects: F110 - Applied chemistry
F200 - Materials science

description: Nanoparticles are increasingly important in biotechnology as they are extensively used as drugdelivery carriers and in biosensors. In both these two contexts, protein-nanoparticle interactionsare often involved. Proteins that are present in body fluids inevitably interact with nanoparticlebased drug carriers and typically surround them forming the so called “protein corona”.Biosensors that are based on nanoparticles often have proteins deliberately attached to theirsurface, for example antibodies that bind specific analytes. The understanding of the assemblymechanisms at the protein-nanoparticle interface and the ability to engineer proteins thatinteract with nanoparticles in the desired way, are therefore two essential requisites for thefuture development of nano-medicines and nano-biosensors.In this work, we focused on the interaction of proteins with gold nanoparticles (GNPs). GNPsare available with a broad range of surface chemistries, suitable for the conjugation of manybiomolecules. Although there are at least three decades of studies on gold colloids withdifferent surface chemistries, there is still quite little known about what are the exact featuresof a protein that determine its adsorption onto gold. We developed methods to study this andapplied them to characterise the adsorption on GNPs of Glutathione-S-Transerase (GST),which was reported previously as a protein that strongly binds gold. We determined its affinityand kinetics of binding and unravelled the mechanism of its thiol-mediated chemisorption. Wefound that GST binds to GNPs even more efficiently than other known gold-binding proteins,such as Bovine Serum Albumin (BSA). We concluded that GST could be considered a veryuseful gold-protein interface, especially considering that GST fusion is routinely used foraffinity purification of recombinant proteins and therefore well established.We also fused self-assembling proteins to GST or chemically cross-linked them to BSA. Thescope was to explore the feasibility of hierarchical and ordered assembly of designer proteinsonto GNPs, with the ultimate goal of providing a convenient tool for modular assembly ofproteins onto nanomaterials. It is known that proteins tend to denature and lose their functionwhen in contact with GNPs, which is not optimal for biosensors or in nanomedicine. We foundthat it is possible to use GST or BSA to form a sacrificial layer on gold, which exposes linked,self-assembling proteins that are able to bind their counterpart, unaffected by the GNP surface.We reported two proof-of-concepts: the first based on mimics of the self-assembling neuronalSNARE proteins and the second based on the pair SpyCatcher/SpyTag, derived fromStreptococcus pyogenes proteins and used in bio-conjugation for their ability to self-catalysethe formation of isopeptidic bonds.We believe that the novel methods and original results presented in this thesis apply to both theunderstanding and the engineering of the protein-nanoparticle interface and will be beneficialfor the broad nanobiotechnology community. In fact, our findings have potential applicationsin a broad range of fields, spanning from the improvement of the circulation life-time ofnanomedicines by preventing the binding of serum protein and opsonisation, to theimprovement of the manufacturing of GNPs-based immune-biosensors such as those used inlateral flow devices.