| The work presented in this thesis describes production of recombinant common carp growth hormone (cGH) in the seed of Brassica napus. The study was carried out in two phases. The first phase consisted of cloning and expression of cGH cDNA in E. coli. This enabled us to verify the GH cDNA and confirm the biological activity of the growth hormone prior to expressing it in plants. Expression in bacteria also provided a source of protein for antibody production and establishment of a rapid bioassay. Since the activity of cGH was tested in goldfish (Carassius auratus ) as a model organism, we cloned and expressed the cDNA encoding the mature goldfish type II growth hormone (gfGH-II) in E. coli for comparative studies. The nucleotide sequence of the gfGH-II cDNA, and its encoded amino acid sequence showed greater than 90% identity to those of other Cyprinid GHs. However, gfGH-II lacked Cys-123 found in the GH of most Cyprinid species. The E. coli-produced recombinant gfGH-II and cGH were sequestered into inclusion bodies, and were solubilized using sodium hydroxide before refolding to restore biological activity. Injection with both GHs significantly increased growth rate in goldfish, while cGH exhibited a greater growth promoting effect than gfGH-II.;The second phase of the work consisted of fusing the cGH cDNA to the 3' end of the 18 kDa A. thaliana oleosin gene, modified previously in our lab to encode a thrombin recognition site at the 3' end of the oleosin coding region, and introduction of the construct into B. napus using Agrobacterium -mediated transformation. The oleosin-cGH fusion was expressed specifically in the seeds of transgenic plants, and was correctly targeted to the oilbodies. Thrombin treatment of the oilbodies resulted in the release of cGH, which was then partially purified by flotation centrifugation. The plant-derived cGH was biologically active as measured by stimulation of the insulin-like growth factor-I (IGF-I) mRNA expression in the liver of goldfish in vitro. Cleaved cGH from B. napus seeds increased the expression of IGF-I mRNA in cultured goldfish liver fractions by a factor of five. Furthermore, injection of the un-cleaved oleosin-cGH stimulated the growth rate of goldfish significantly (p < 0.05). A novel finding of this work was that cGH exhibited biological activity as a fusion protein. All these results contrast with previous reports, which suggested that plants, and especially seeds, were poor hosts for the production of recombinant growth hormone. |
