Self-assembly of biomaterials and bioimaging using novel peptides
thesisposted on 28.03.2022, 17:13 authored by Andrew Care
Protein immobilisation on solid matrices commonly relies on non-specific adsorption or the reaction between chemical groups within proteins and those on the matrix surface. In both cases, proteins attach to the surfaces in random orientations that may cause a reduction or loss of biological activity. Some peptides are capable of directing the immobilisation and orientation of proteins on solid surfaces without impeding protein function. They can recognise selectively and bind to a diverse range of inorganic substrates, for example, metals, materials containing carbon and polymers. In this thesis I present a robust and versatile bioconjugation system that circumvents conventional chemical methods. This technology is based on a unique peptide linker sequence that displays high affinity towards materials that contain silica. The linker sequence can be fused directly to the sequence of a protein of interest using genetic engineering techniques and produced in Escherichia coli. The resulting recombinant fusion protein (Linker-Protein) exhibits strong affinity to a range of materials that contain silica, including synthetic silica and zeolites. Several applications of this technology are presented in this thesis. The filamentous fungus Trichoderma reesei was tested as an alternative host to achieve higher yields and extracellular production of a linker-enzyme complex. However, the linker and several derivatives were rapidly degraded by T. reesei proteinases and further modification of the sequence is required by a directed evolution technique such as phage display. In a further application, the linker was incorporated into Streptococcus Protein G, an antibody-binding protein. The resulting Linker-Protein G (LPG) was able to direct the attachment of antibodies onto silica-coated magnetic particles. The biofunctionalised particles were used for the antibody-mediated binding and recovery of different cell types for rapid, simple visualisation and identification by microscopy. The application of LPG was extended further by the incorporation of luminescent lanthanide chelates. The Luminescence-Activating LPG (LA-LPG) was shown to impart strong luminescence to antibodies within seconds and represents a novel indirect detection reagent for time-gated luminescence bioimaging.