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Reason: Under embargo until Sep 2017. After this date a copy can be supplied under Section 51(2) of the Australian Copyright Act 1968 by submitting a document delivery request through your library
Expression and characterisation of plant-made recombinant hEGF
thesisposted on 2017-01-24, 00:55 authored by Thomas, David Rhys
Human epidermal growth factor (hEGF) is a powerful mitogen first characterised in the early 1970’s. Since its initial discovery, hEGF has been shown to enhance the healing of a wide range of injuries, including burns, cuts, diabetic ulcers and gastric ulcers. However, despite its clinical value hEGF is only consistently used for for the treatment of chronic diabetic ulcers. Currently, the high cost of hEGF combined with the large doses needed for clinical effect, are significant hurdles to its widespread use. Human epidermal growth factor is a 53 amino acid protein containing three disulphide bonds. These bonds make production of hEGF through traditional E. coli methods inefficient, as prokaryotes are naturally unable to produce disulphide bonds. This has led to the exploration of alternative expression platforms, including the use of plants. While plants are able to produce correctly folded hEGF, the concentrations produced to date are too low to be of commercial value. This thesis attempts to address the issues currently preventing the widespread use of hEGF. The first approach taken was to optimise the expression of hEGF when transiently expressed in Nicotiana benthamiana plants. Through a variety of techniques, including targeting hEGF to various sub-cellular compartments, co-expressing a silencing suppressor and codon optimisation of the hEGF DNA sequence, yield was increased 10-fold above initial concentrations. A second approach to increase hEGF expression was to target hEGF to multiple sub-cellular compartments simultaneously from bicistronic mRNA. While these constructs were found to be appropriately processed to produce mature hEGF, they were not able to increase the accumulation of hEGF compared to targeting the vacuole alone. This is thought to be due to an increase in the amount of hEGF passing through the ER activating the unfolded protein response (UPR), reducing the cells rate of protein synthesis. The final approach in this study attempted to decrease the amount of hEGF needed for activity by fusing the collagen binding domain (CBD) of human bone sialoprotein to hEGF. While the CBD was found to be removed from the majority of hEGF peptides, there was still detectable collagen binding in samples. When cells were grown on a collagen substrate, hEGF with a CBD enhanced cell growth to a greater extent than soluble hEGF peptide alone. This thesis found that recombinant hEGF expression can be enhanced by a variety of optimisations, however, high hEGF yields may be limited by the activity of the unfolded protein response in cells. Adding a CBD to hEGF increased the activity of the bound protein compared to soluble hEGF, making the fusion protein potentially valuable for therapeutic use. The implications of this study as well as future areas for research are also discussed.