No description available.

Bibliography: leaves 215-236. / x, 249 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Scale-up of a biochemical process involving expression of an Insulin-like Growth Factor-I analogue (LongR3IGF-I) as inclusion bodies within the bacterium Escherichia coli has been investigated. The principal focus was directed to the operation of refolding wherein the biological potency of the protein is imparted. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemical Engineering, 1998?

Bibliography: leaves 215-236. / x, 249 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Scale-up of a biochemical process involving expression of an Insulin-like Growth Factor-I analogue (LongR3IGF-I) as inclusion bodies within the bacterium Escherichia coli has been investigated. The principal focus was directed to the operation of refolding wherein the biological potency of the protein is imparted. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemical Engineering, 1998?

Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available in print.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 128-142).

published_or_final_version / abstract / Physiology / Master / Master of Philosophy

by Sharon, Chui-Wah Luk. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 128-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Methylobacterium extorquens ATCC55366 is an interesting candidate for large-scale production of recombinant proteins. Development and optimization of this recombinant expression system were done using the green fluorescent protein (GFP) gene cloned into expression vectors (pRK310 and pCM110) as model systems. Selection of efficient GFP-expressing clones, long-term production stability without selection in flasks, effects of selection, oxygen and methanol supplies, were studied during fed-batch fermentations in a 20-l bioreactor. Sequential batch-culture cultivations in shake flasks showed that specific GFP production was constant in the presence of tetracycline. However, the GFP production decreased in the absence of this selective pressure. In fed-batch fermentations of recombinant M. extorquens ATCC 55366 (pMxaF-GFP), overall GFP yields (≈70 mg/g; GFP/cell dry weight) were not affected by the presence or absence of tetracycline, nor by oxygen and methanol concentration oscillations. The cry1Aa gene from Bacillus thuringiensis kurstaki NRD-12 was cloned in pCM110 and then transformed into M. extorquens. Heterologous expression of the cry1Aa gene in M. extorquens AM1 and ATCC 55366 was detected by immunoblot analyses. This study suggests that M. extorquens can be used as a valuable expression system for intracellular recombinant protein production.

Many proteins with special properties have been identified in aquatic organisms. Due to their peculiarity, some of these biomolecules could be used advantageously for some industrial and health care applications. However, the preparation of these biochemicals from its original source is highly impractical and generally unfeasible for commercial purposes. Nevertheless, the advances in DNA manipulation open the possibility of producing the recombinant proteins in different organisms to allow a viable production and extraction on a commercial scale. Among such particular proteins, the cunner-fish trypsin (CFT) is an enzyme with a high potential for exploitation by the food processing industry. The CFT is a cold adapted protease that has a proven ability to inactivate undesirable endogenous enzymes during the processing of some foodstuffs. Thus, the CFT gene was characterized and cloned in E. coli and Pichia pastoris for over-expression. A cDNA library from pancreatic mRNA was screened using a salmon trypsin cDNA probe. Positive clones harboring a cDNA insert of the predicted size were sequenced and a full-length cDNA was obtained that contained an ORF encoding the zymogen and a signal peptide. Multiple alignment of the gene sequence showed 85 to 90 per cent identities with other fish trypsins and the deduced amino acid sequence for the mature enzyme exhibited the entire characteristic features observed in trypsins. E. coli expressed the recombinant CFT (rCFT) at levels higher than 20% in the form of insoluble aggregates, while P. pastoris showed expression levels below 1%. Despite the low expression, the yeast rCFT appeared to be correctly folded and could be partially purified by immobilized nickel affinity chromatography. Trypsin-specific activity was confirmed in the purified rCFT after digestion with bovine enterokinase.