| Monitoring of transcript regulation and protein production related stress responses in Pichia pastoris secreting Fab antibody fragments | |
Abstract/OtherAbstract
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VTT Symposium 242. International Specialised Symposium on Yeasts ISSY25. Systems Biology of Yeasts - from Models to Applications, 105, The rapid transcriptional profiling method VTT-TRAC has been applied to monitor the levels of a subset of mRNAs coding for UPR-regulated and stress-connected genes in chemostat cultivations of a P. pastoris strain secreting the 2F5 antibody fragment. Specific marker genes have been chosen to deliver insights into the general physiological status of the cells under production conditions (including growth, protein synthesis, oxygen and nutrient limitation responses) with the main focus on secretion stress connected genes (UPR, ERAD, posttranslational processing). TRAC analysis of shake flask cultivations of P. pastoris strains co-overexpressing S. cerevisiae UPR-transcription factor Hac1p was applied to identify UPR-targets in P. pastoris. Consequently, the induction of UPR-target genes due to heterologous protein production could be shown for the first time in P. pastoris. Overexpression of S. cerevisiae protein disulfide isomerase (PDI1) could not diminish UPR burden. Most of the investigated genes seem to be unaffected by PDI1 overexpression at a transcriptional level, although 2F5 Fab secretion rates are enhanced under these conditions. As product formation is known to be strongly dependent on cultivation conditions, the influence of different cultivation temperatures has been analysed. Transcript formation rates of the two respective product genes (for Fab light chain and heavy chain mRNA) have been set in correlation to the mRNA levels of folding related genes such as KAR2 and PDI1, and additionally to the specific product formation rate of secreted Fab. Interestingly, although the transcriptional levels of our product genes were reduced at lower temperature, specific productivity of the 2F5 Fab protein was significantly increased. Thus it is tempting to speculate that at lower temperature a reduced amount of folding stress is imposed on the cells, consequently leading to a higher rate of correctly folded product. Also the chaperon KAR2/BiP, which is commonly seen as a marker of unfolded protein stress appeared among the genes downregulated at lower temperature, while the transcription of genes coding for elements of the vesicular transport system is enhanced. Additionally, the levels of intracellularly retained antibody fragments and the UPR marker protein BiP (Kar2) were analyzed by immunofluorescence and flow cytometry. Conclusions will be drawn regarding the regulation patterns of the analyzed marker genes during the adaptation to different growth conditions, and their connection to product formation and secretion will be discussed., The rapid transcriptional profiling method VTT-TRAC has been applied to monitor the levels of a subset of mRNAs coding for UPR-regulated and stress-connected genes in chemostat cultivations of a P. pastoris strain secreting the 2F5 antibody fragment. Specific marker genes have been chosen to deliver insights into the general physiological status of the cells under production conditions (including growth, protein synthesis, oxygen and nutrient limitation responses) with the main focus on secretion stress connected genes (UPR, ERAD, posttranslational processing). TRAC analysis of shake flask cultivations of P. pastoris strains co-overexpressing S. cerevisiae UPR-transcription factor Hac1p was applied to identify UPR-targets in P. pastoris. Consequently, the induction of UPR-target genes due to heterologous protein production could be shown for the first time in P. pastoris. Overexpression of S. cerevisiae protein disulfide isomerase (PDI1) could not diminish UPR burden. Most of the investigated genes seem to be unaffected by PDI1 overexpression at a transcriptional level, although 2F5 Fab secretion rates are enhanced under these conditions. As product formation is known to be strongly dependent on cultivation conditions, the influence of different cultivation temperatures has been analysed. Transcript formation rates of the two respective product genes (for Fab light chain and heavy chain mRNA) have been set in correlation to the mRNA levels of folding related genes such as KAR2 and PDI1, and additionally to the specific product formation rate of secreted Fab. Interestingly, although the transcriptional levels of our product genes were reduced at lower temperature, specific productivity of the 2F5 Fab protein was significantly increased. Thus it is tempting to speculate that at lower temperature a reduced amount of folding stress is imposed on the cells, consequently leading to a higher rate of correctly folded product. Also the chaperon KAR2/BiP, which is commonly seen as a marker of unfolded protein stress appeared among the genes downregulated at lower temperature, while the transcription of genes coding for elements of the vesicular transport system is enhanced. Additionally, the levels of intracellularly retained antibody fragments and the UPR marker protein BiP (Kar2) were analyzed by immunofluorescence and flow cytometry. Conclusions will be drawn regarding the regulation patterns of the analyzed marker genes during the adaptation to different growth conditions, and their connection to product formation and secretion will be discussed. |
Authors
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Gasser, B., Maurer, M., Sauer, M., Saloheimo, Markku, Rautio, Jari, Penttilä, Merja, Mattanovich, D. |
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Publisher : VTT Type : text Format : application/pdf |
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2006 |
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Copyright VTT Technical Research Centre of Finland. Full text may not be reproduced, republished, stored, distributed, transmitted, altered or resold except as follows: Full text may be downloaded, held and displayed for private study or research and single copies may be printed out for private study or research. In all citations the source and the copyright holder must be acknowledged. The documents are provided "as is" and "as available" basis. No warranty of kind, either express or implied including but not limited to warranties of title or non-infringement or implied warranties of merchantability or fitness for a particular purpose, is made in relation to the availability, accuracy, reliabi lity or content of these pages. |
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Languages : eng |
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