Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.579631
Title: Protein scaffolds for cell culture
Author: Machado Roque, Ana Isabel
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2013
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Abstract:
We report here the design, purification and structural characterization of a new protein scaffold for cell culture. Prior studies in our group revealed the structure of the bacterial protein Caf1 to be flexible protein nanofibres, up to 1.5 μm. The existing Cafl expression system was cumbersome and difficult to mutate, we have now produced a system containing the caf operon which allows for the incorporation of specific peptide motifs. The small peptide, RGDS from fibronectin was inserted into 5 different surface loops of Caf1. The Caf1 mutants were expressed and purified and a structural characterization by biophysical methods was conducted. This revealed permissive sites into which new motifs can be inserted. The characterised proteins were sterilised and used to coat 96 well plates for cell culture. In this study we used mammalian cell lines such as 3T3 fibroblasts, PC12 neuronal cells and primary osteoblasts to understand how they behave in the presence of this biomaterial, in particular the formation of focal adhesions, changes in cytoskeleton rearrangement and nuclear and cell morphology. The controlled engineering of sites within the polymer allowed us to study their implication in cell attachment, survival and proliferation. Our preliminary results have shown that cells interact poorly with the unmodified protein e.g. without any motif associated. This reveals that the polymer is inert and does not influence cell growth by itself. In contrast, the incorporation of RGDS, can invert the scenario of cell growth; promoting cell attachment, survival and proliferation. In a second stage of the project we designed a separate compatible plasmid encoding caf1 gene and used it with the previous plasmid to co-express hybrid Caf1 polymers. The long fibres can also be crosslinked with a non-toxic and non-immunogenic chemical compound – NHS-PEG. Thus a protein hydrogel composed of interchangeable folding units which can be used to incorporate different cell interacting peptide motifs. It is robust and, in the unmodified state highly protease resistant. Future studies will elucidate the versatility and potentiality for this peptide hydrogel in stem cell differentiation.
Supervisor: Not available Sponsor: Medical Research Council (MRC) ; Orla Proteins and Technologies
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.579631  DOI: Not available
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