The work presented in this thesis describes the production and characterisation of recombinant streptavidin-cytokine fusion proteins. Immunoadjuvants can augment the weak or non-existent antitumour response of a patient with cancer to immunotherapy. The response of breast cancer patients to cancer vaccines directed against tumour associated antigens, such as HER2/neu, is often weak and it has been reported that additional immunostimulation, such as that provided by an immunoadjuvant, is required in order to achieve effective therapeutic results. One approach to the development of novel immunoadjuvants is the production of fusion proteins which are designed to perform the dual functions of general activation of the immune system as well as directing the immune response specifically towards a TH1-type response, which is widely regarded as the optimal patient response for cancer immunotherapy.Streptavidin is a bacterial protein and an immunogenic molecule that induces an unspecific immune response. The cytokines IL-2 and IL-18 both promote TH1-type responses; IL-2 is already in clinical use as a cancer therapy, while investigation of the role of IL-18 as an immunotherapy is ongoing.A strategy was devised for the production of novel recombinant fusion proteins designed for use as immunoadjuvants; these proteins comprised an N-terminal truncated streptavidin core protein sequence and a C-terminal cytokine, specifically either IL-2 or IL-18, separated by a short polypeptide linker region. Molecular cloning techniques were used to generate DNA expression constructs encoding the recombinant fusion proteins which were expressed in an inducible bacterial system using plasmid expression vector pCR(R)T7/NT-TOPO(R). The expressed recombinant proteins were found to accumulate within insoluble bacterial inclusion bodies and protocols were developed and optimised for the isolation and solubilization of these proteins. Protein solubilization required the use of buffers at high pH (pH 12.5) which resulted in disrupting protein structural integrity; a pulsed dilution method was subsequently employed to achieve refolding of the proteins prior to further analysis.Characterization of fusion proteins STV/IL-2 (streptavidin-IL-2) and STV/IL-18 (streptavidin-IL-18) was conducted using native and dissociating PAGE and Western analysis. Antibody binding studies provided preliminary confirmation of the identity of the STV/IL-2 fusion protein. Similar studies to characterize STV/IL-18 were initially encouraging but proved inconclusive and further analysis of this protein is required.These initial investigations have validated this approach using the expression systems established here for the production of recombinant streptavidin-cytokine fusion proteins. A number of issues to be addressed have been highlighted regarding problems encountered with protein yields, solubilisation and maintenance of structural integrity. It is therefore concluded that further modification and optimisation of the expression system and protein isolation procedures employed is necessary to provide an appropriate system for the production of these fusion proteins; this will subsequently permit the investigation of their potential for use in therapeutic applications.
Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:494851 |
Date | January 2008 |
Creators | Henderson, Rosalind |
Contributors | Dyer, Charlotte ; Greenman, John |
Publisher | University of Hull |
Source Sets | Ethos UK |
Detected Language | English |
Type | Electronic Thesis or Dissertation |
Source | http://hydra.hull.ac.uk/resources/hull:1606 |
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