Monitoring of transcript regulation and protein production related stress responses in Pichia pastoris secreting Fab antibody fragments

B. Gasser, M. Maurer, M. Sauer, Markku Saloheimo, Jari Rautio, Merja Penttilä, D. Mattanovich

Research output: Chapter in Book/Report/Conference proceedingConference abstract in proceedingsScientific

11 Citations (Scopus)

Abstract

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.
Original languageEnglish
Title of host publicationInternational Specialised Symposium on Yeasts ISSY25
Subtitle of host publicationSystems Biology of Yeasts - from Models to Applications
PublisherVTT Technical Research Centre of Finland
Pages105
ISBN (Electronic)951-38-6308-5
ISBN (Print)951-38-6307-7
Publication statusPublished - 2006
EventInternational Specialised Symposium on Yeasts, ISSY 25 - Espoo, Finland
Duration: 18 Jun 200621 Jun 2006

Publication series

SeriesVTT Symposium
Number242
ISSN0357-9387

Conference

ConferenceInternational Specialised Symposium on Yeasts, ISSY 25
Abbreviated titleISSY 25
CountryFinland
CityEspoo
Period18/06/0621/06/06

Fingerprint

Pichia pastoris
stress response
antibodies
monitoring
genes
proteins
secretion
liquid state fermentation
temperature
protein disulfide-isomerase
genetic markers
physiological state
fluorescent antibody technique
flow cytometry
transcription factors
transcription (genetics)
protein synthesis
cells
oxygen

Cite this

Gasser, B., Maurer, M., Sauer, M., Saloheimo, M., Rautio, J., Penttilä, M., & Mattanovich, D. (2006). Monitoring of transcript regulation and protein production related stress responses in Pichia pastoris secreting Fab antibody fragments. In International Specialised Symposium on Yeasts ISSY25: Systems Biology of Yeasts - from Models to Applications (pp. 105). [P52] VTT Technical Research Centre of Finland. VTT Symposium, No. 242
Gasser, B. ; Maurer, M. ; Sauer, M. ; Saloheimo, Markku ; Rautio, Jari ; Penttilä, Merja ; Mattanovich, D. / Monitoring of transcript regulation and protein production related stress responses in Pichia pastoris secreting Fab antibody fragments. International Specialised Symposium on Yeasts ISSY25: Systems Biology of Yeasts - from Models to Applications. VTT Technical Research Centre of Finland, 2006. pp. 105 (VTT Symposium; No. 242).
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abstract = "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.",
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Gasser, B, Maurer, M, Sauer, M, Saloheimo, M, Rautio, J, Penttilä, M & Mattanovich, D 2006, Monitoring of transcript regulation and protein production related stress responses in Pichia pastoris secreting Fab antibody fragments. in International Specialised Symposium on Yeasts ISSY25: Systems Biology of Yeasts - from Models to Applications., P52, VTT Technical Research Centre of Finland, VTT Symposium, no. 242, pp. 105, International Specialised Symposium on Yeasts, ISSY 25 , Espoo, Finland, 18/06/06.

Monitoring of transcript regulation and protein production related stress responses in Pichia pastoris secreting Fab antibody fragments. / Gasser, B.; Maurer, M.; Sauer, M.; Saloheimo, Markku; Rautio, Jari; Penttilä, Merja; Mattanovich, D.

International Specialised Symposium on Yeasts ISSY25: Systems Biology of Yeasts - from Models to Applications. VTT Technical Research Centre of Finland, 2006. p. 105 P52 (VTT Symposium; No. 242).

Research output: Chapter in Book/Report/Conference proceedingConference abstract in proceedingsScientific

TY - CHAP

T1 - Monitoring of transcript regulation and protein production related stress responses in Pichia pastoris secreting Fab antibody fragments

AU - Gasser, B.

AU - Maurer, M.

AU - Sauer, M.

AU - Saloheimo, Markku

AU - Rautio, Jari

AU - Penttilä, Merja

AU - Mattanovich, D.

PY - 2006

Y1 - 2006

N2 - 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.

AB - 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.

M3 - Conference abstract in proceedings

SN - 951-38-6307-7

T3 - VTT Symposium

SP - 105

BT - International Specialised Symposium on Yeasts ISSY25

PB - VTT Technical Research Centre of Finland

ER -

Gasser B, Maurer M, Sauer M, Saloheimo M, Rautio J, Penttilä M et al. Monitoring of transcript regulation and protein production related stress responses in Pichia pastoris secreting Fab antibody fragments. In International Specialised Symposium on Yeasts ISSY25: Systems Biology of Yeasts - from Models to Applications. VTT Technical Research Centre of Finland. 2006. p. 105. P52. (VTT Symposium; No. 242).