Abstract
The work is part of a EUROSCOPE programme project Genophys, in which the aim is to compare the physiology of heterologous protein production in different microbial production hosts using a common model protein and applying genome-wide analysis methods to study the cellular events at a selected set of cultivation conditions.
We have carried out transcriptome and proteome analysis of a Trichoderma reesei strain producing antibody 3H6 Fab-fragment and a control strain expressing only the selection marker gene amdS, and analysed the effects of production of the hetereologous protein as well as cultivation temperature on the cellular responses. For the analyses, the strains were cultivated in lactose-limited chemostats at 21.5°C, 24°C and 28°C. The dilution rate used in the cultivations, 0.03/h, has been previously shown to be optimal for protein production in similar type of chemostat cultures of T. reesei. The lower temperatures used in the study, favoured protein production by both strains. In the Fab producing strain, both the total protein production into the culture medium as well as Fab production were the highest at 21.5°C, the values obtained at 24°C being close to the ones obtained at 21.5°C. In the control strain the highest protein production was at 24°C. Comparison of differentially expressed genes between the strains showed relatively few genes affected by the heterologous gene expression, whereas comparison of the cultures at different temperatures revealed a large number of genes with altered expression level. However, the proteome analysis of the cultures using the 2D DIGE method indicated also stress responses to the heterologous protein production. The analysis showed differences especially in the intensity of protein spots corresponding to different type of heat shock proteins (ER, mitochondrial, cytoplasmic, ribosome-associated HSPs), actin and tubulin assembly factors, and proteins related to ER-associated protein degradation.
We have carried out transcriptome and proteome analysis of a Trichoderma reesei strain producing antibody 3H6 Fab-fragment and a control strain expressing only the selection marker gene amdS, and analysed the effects of production of the hetereologous protein as well as cultivation temperature on the cellular responses. For the analyses, the strains were cultivated in lactose-limited chemostats at 21.5°C, 24°C and 28°C. The dilution rate used in the cultivations, 0.03/h, has been previously shown to be optimal for protein production in similar type of chemostat cultures of T. reesei. The lower temperatures used in the study, favoured protein production by both strains. In the Fab producing strain, both the total protein production into the culture medium as well as Fab production were the highest at 21.5°C, the values obtained at 24°C being close to the ones obtained at 21.5°C. In the control strain the highest protein production was at 24°C. Comparison of differentially expressed genes between the strains showed relatively few genes affected by the heterologous gene expression, whereas comparison of the cultures at different temperatures revealed a large number of genes with altered expression level. However, the proteome analysis of the cultures using the 2D DIGE method indicated also stress responses to the heterologous protein production. The analysis showed differences especially in the intensity of protein spots corresponding to different type of heat shock proteins (ER, mitochondrial, cytoplasmic, ribosome-associated HSPs), actin and tubulin assembly factors, and proteins related to ER-associated protein degradation.
| Original language | English |
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| Publication status | Published - 2010 |
| MoE publication type | Not Eligible |
| Event | 10th European Conference on Fungal Genetics - Amsterdam, Netherlands Duration: 29 Mar 2010 → 1 Apr 2010 |
Conference
| Conference | 10th European Conference on Fungal Genetics |
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| Country/Territory | Netherlands |
| City | Amsterdam |
| Period | 29/03/10 → 1/04/10 |