The development of a system to facilitate the stable expression of mammalian proteins in the filamentous fungus 'Aspergillus oryzae'
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Human interleukin 6 (hIL6) is a multifunctional cytokine effecting the function and proliferation of many cell types. The further understanding of hIL6 and its possible medical applications rely on the availability of this protein. The filamentous fungus Aspergillus oryzae is an important industrial organism and is used for the large scale production of many enzymes. As this fungus has an impressive secretory output it was decided to attempt to produce hIL6 in this organism. The initial work was based on the development of a gene transfer system for A. oryzae in order that the hIL6 gene could be introduced to the organism. A transformation system based on the homologous nitrate reductase gene is described. This system yielded up to 800 transformants per µg plasmid DNA. Additionally, the adaptation of the transformation system based on the A. nidulans anuiS gene and the use of other transformation systems is reported. In order to ensure that the hIL6 gene was efficiently transcribed it was considered important that homologous control regions from highly produced and regulated A. oryzae genes were linked to the hIL6 gene. Therefore the genes encoding glucoamylase and a-amylase were isolated from A. oryzae. A method for the purification of A. oryzae Si nuclease is described and the amino acid sequence of the N terminus is reported. A. oryzae produces large amounts of extracellular proteases, a feature unlikely to be attractive in a heterologous host. Therefore the production of protease production in A. oryzae was studied. A method is described for the selection of protease deficient mutants. Using this method two protease mutants were isolated and these have been characterized. One mutation designated prtA2 protects against the degradation of hJL6 in vitro. The A. oryzae alkaline protease gene was isolated and mutagenised and the attempts made to produce specific protease mutant by reverse genetics are described. A system is described where by A. oryzae can be engineered to produce relatively high levels of hIL6. Using gene fusion constructs transformants producing in the range of 1 mg per Litre have been isolated. This system is heterologous, recommendations for increasing hIL6 production are included.
Thesis, PhD Doctor of Philosophy
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