Amyloid fibrils are an insoluble, highly ordered, fibrous protein structure, which have increasingly been recognised as having bionanotechnology applications. Their ability to selfassemble allows a bottom-up approach to material design. Their nanometre dimensions affords them a high surface-to-volume ratio and their proteinaceous building blocks from which they are assembled allow for decoration with biomolecules and chemicals through amino acid residues. Amyloid fibrils are therefore a potential nanoscaffold for immobilisation of biomolecules.
Immobilisation offers a solution to the problems associated with the use of enzymes in in vitro applications, by increasing their stability, reusability, and in some cases, enhancing catalytic activity. Nanosupports offer a high surface-to-volume ratio compared to classical planar 2-D supports, potentially affording them dramatic increases in immobilisation capacity.
To investigate the potential of amyloid fibrils as a novel nanoscaffold, organophosphate hydrolase (OPH), cytochrome P450BM3 (P450BM3), green fluorescent protein (GFP), tobacco
etch virus protease (TEV), and glucose oxidase (GOD) were immobilised in solution to the model amyloid fibril forming protein, bovine insulin. Covalently immobilised OPH was found to have a ~300 % increase in relative thermostability at 40 and 50 °C. P450BM3 was not successfully immobilised in its active state, most likely due to unfolding of the enzyme on the amyloid fibril surface. Covalently immobilised GFP retained full fluorescence and acted as a fluorescent protein tag. TEV was shown to have a physical interaction with the nanoscaffold and retain activity. GOD was immobilised and retained activity. Although not all proteins retained activity, a range of different protein structures were successfully immobilised onto the insulin amyloid fibril nanoscaffold. Attachment to the crystallin amyloid fibril nanoscaffold remains a work in progress due to the complexities associated with post-translational modifications of these fibrils. Crystallin amyloid fibrils were assembled on a surface for the first time. Their surface assembled structure was found to resemble spherulites, not previously seen before with crystallin amyloid fibrils.
Bovine insulin amyloid fibrils were assembled on the surface of glass beads to increase the available surface area for biomolecule immobilisation. The surface assembled bovine insulin nanoscaffold was first functionalised with GOD, demonstrating that the nanoscaffold provides more surface area for biomolecule immobilisation, although in this case the increase was limited due to high non-specific binding of GOD to the unmodified glass
surface. GFP was successfully employed as a fluorescent protein tag to assess the degree of nanoscaffold coverage, confirming the nanoscaffold affords the glass bead a greater surface area. Moreover, a reusable immobilised TEV protease-bead system was developed that was able to sequentially cleave the poly-histidine tags of three different proteins.
In conclusion, bovine insulin amyloid fibrils have been shown to be a versatile nanoscaffold for the immobilisation of a range of biomolecules. The surface characteristics of the nanoscaffold allows for both covalent and physical immobilisation of biomolecules. Thus, amyloid fibrils have exciting potential in the creation of novel bionanotechnologies.
| Identifer | oai:union.ndltd.org:canterbury.ac.nz/oai:ir.canterbury.ac.nz:10092/6925 |
| Date | January 2012 |
| Creators | Raynes, Jared Kenneth |
| Publisher | University of Canterbury. Biological Sciences |
| Source Sets | University of Canterbury |
| Language | English |
| Detected Language | English |
| Type | Electronic thesis or dissertation, Text |
| Rights | Copyright Jared Kenneth Raynes, http://library.canterbury.ac.nz/thesis/etheses_copyright.shtml |
| Relation | NZCU |
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