Analysis of Heterologous Protein Production in Defined Recombinant Aspergillus awamori Strains

Robin J. Gouka, Peter J. Punt, Johanna (H.) G. M. Hessing and Cees A.M.J.J. van den Hondel

Author address: 

TNO Nutrition and Food Research Institute, Department of Molecular Genetics and Gene Technology, PO Box 5815, 2280 HV Rijswijk, the Netherlands

Abstract: 

Filamentous fungi, especially members of the genus Aspergillus, are able to secrete large amounts of homologous proteins into the medium which makes them attractive as a host for protein production. In contrast, heterologous proteins are very poorly produced and secreted. The objective of our research is to obtain more insight in the parameters that influence heterologous protein production in Aspergillus awamori. To investigate this, a systematic analysis was carried out in which the expression levels of a number of different fungal and non-fungal genes were analyzed. This method is based on the single copy integration of different expression cassettes at the pyrG locus of A. awamori. Differences in expression mainly occured at the steady state mRNA level, varying from high mRNA levels for genes of fungal origin to low levels for genes of non-fungal origin. With one gene, encoding plant Cyamopsis tetragonoloba -galactosidase, no full length mRNA could be detected. With RT-PR and nuclear run-on transcription assays it could be demonstrated that incorrect processing of full length mRNA was probably occurring, resulting in the lack of about 900 nt in the mRNA. By changing the DNA sequence of the gene improved levels of full length mRNA could be obtained. In most cases the protein levels corresponded to the amount expected on basis of the mRNA levels. Only in the case of human interleukin-6, relatively high mRNA levels were obtained, whereas, only very low amounts of protein could be detected.To further investigate the problems observed for plant agalactosidase and human IL6, gene fusions with the A. niger glucoamylase gene (glaA) were constructed. Data on improved mRNA and protein levels will be presented.
1996

abstract No: 

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Full conference title: 

3rd EUROPEAN CONFERENCE ON FUNGAL GENETICS
    • ECFG 3rd (1996)