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The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins

Mouna Guerfal UGent, Stefan Ryckaert, Pieter Jacobs, Paul Ameloot UGent, Kathleen Van Craenenbroeck UGent, Riet De Rycke UGent and Nico Callewaert UGent (2010) MICROBIAL CELL FACTORIES. 9.
abstract
Background: The unfolded protein response (UPR) in eukaryotes upregulates factors that restore ER homeostasis upon protein folding stress and in yeast is activated by a non-conventional splicing of the HAC1 mRNA. The spliced HAC1 mRNA encodes an active transcription factor that binds to UPR-responsive elements in the promoter of UPR target genes. Overexpression of the HAC1 gene of S. cerevisiae can reportedly lead to increased production of heterologous proteins. To further such studies in the biotechnology favored yeast Pichia pastoris, we cloned and characterized the P. pastoris HAC1 gene and the splice event. Results: We identified the HAC1 homologue of P. pastoris and its splice sites. Surprisingly, we could not find evidence for the non-spliced HAC1 mRNA when P. pastoris was cultivated in a standard growth medium without any endoplasmic reticulum stress inducers, indicating that the UPR is constitutively active to some extent in this organism. After identification of the sequence encoding active Hac1p we evaluated the effect of its overexpression in Pichia. The KAR2 UPR-responsive gene was strongly upregulated. Electron microscopy revealed an expansion of the intracellular membranes in Hac1p-overexpressing strains. We then evaluated the effect of inducible and constitutive UPR induction on the production of secreted, surface displayed and membrane proteins. Wherever Hac1p overexpression affected heterologous protein expression levels, this effect was always stronger when Hac1p expression was inducible rather than constitutive. Depending on the heterologous protein, co-expression of Hac1p increased, decreased or had no effect on expression level. Moreover, alpha-mating factor prepro signal processing of a G-protein coupled receptor was more efficient with Hac1p overexpression; resulting in a significantly improved homogeneity. Conclusions: Overexpression of P. pastoris Hac1p can be used to increase the production of heterologous proteins but needs to be evaluated on a case by case basis. Inducible Hac1p expression is more effective than constitutive expression. Correct processing and thus homogeneity of proteins that are difficult to express, such as GPCRs, can be increased by co-expression with Hac1p.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
YEAST, ACTIVATION, INDUCTION, IRE1, REVEALS, EXPRESSION
journal title
MICROBIAL CELL FACTORIES
Microb. Cell. Fact.
volume
9
article_number
49
pages
12 pages
Web of Science type
Article
Web of Science id
000280174400001
JCR category
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
JCR impact factor
4.544 (2010)
JCR rank
21/158 (2010)
JCR quartile
1 (2010)
ISSN
1475-2859
DOI
10.1186/1475-2859-9-49
language
English
UGent publication?
yes
classification
A1
copyright statement
I have retained and own the full copyright for this publication
id
1019468
handle
http://hdl.handle.net/1854/LU-1019468
date created
2010-08-09 12:26:20
date last changed
2012-06-26 14:32:05
@article{1019468,
  abstract     = {Background: The unfolded protein response (UPR) in eukaryotes upregulates factors that restore ER homeostasis upon protein folding stress and in yeast is activated by a non-conventional splicing of the HAC1 mRNA. The spliced HAC1 mRNA encodes an active transcription factor that binds to UPR-responsive elements in the promoter of UPR target genes. Overexpression of the HAC1 gene of S. cerevisiae can reportedly lead to increased production of heterologous proteins. To further such studies in the biotechnology favored yeast Pichia pastoris, we cloned and characterized the P. pastoris HAC1 gene and the splice event.
Results: We identified the HAC1 homologue of P. pastoris and its splice sites. Surprisingly, we could not find evidence for the non-spliced HAC1 mRNA when P. pastoris was cultivated in a standard growth medium without any endoplasmic reticulum stress inducers, indicating that the UPR is constitutively active to some extent in this organism. After identification of the sequence encoding active Hac1p we evaluated the effect of its overexpression in Pichia. The KAR2 UPR-responsive gene was strongly upregulated. Electron microscopy revealed an expansion of the intracellular membranes in Hac1p-overexpressing strains. We then evaluated the effect of inducible and constitutive UPR induction on the production of secreted, surface displayed and membrane proteins. Wherever Hac1p overexpression affected heterologous protein expression levels, this effect was always stronger when Hac1p expression was inducible rather than constitutive. Depending on the heterologous protein, co-expression of Hac1p increased, decreased or had no effect on expression level. Moreover, alpha-mating factor prepro signal processing of a G-protein coupled receptor was more efficient with Hac1p overexpression; resulting in a significantly improved homogeneity.
Conclusions: Overexpression of P. pastoris Hac1p can be used to increase the production of heterologous proteins but needs to be evaluated on a case by case basis. Inducible Hac1p expression is more effective than constitutive expression. Correct processing and thus homogeneity of proteins that are difficult to express, such as GPCRs, can be increased by co-expression with Hac1p.},
  articleno    = {49},
  author       = {Guerfal, Mouna and Ryckaert, Stefan and Jacobs, Pieter and Ameloot, Paul and Van Craenenbroeck, Kathleen and De Rycke, Riet and Callewaert, Nico},
  issn         = {1475-2859},
  journal      = {MICROBIAL CELL FACTORIES},
  keyword      = {YEAST,ACTIVATION,INDUCTION,IRE1,REVEALS,EXPRESSION},
  language     = {eng},
  pages        = {12},
  title        = {The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins},
  url          = {http://dx.doi.org/10.1186/1475-2859-9-49},
  volume       = {9},
  year         = {2010},
}

Chicago
Guerfal, Mouna, Stefan Ryckaert, Pieter Jacobs, Paul Ameloot, Kathleen Van Craenenbroeck, Riet De Rycke, and Nico Callewaert. 2010. “The HAC1 Gene from Pichia Pastoris: Characterization and Effect of Its Overexpression on the Production of Secreted, Surface Displayed and Membrane Proteins.” Microbial Cell Factories 9.
APA
Guerfal, M., Ryckaert, S., Jacobs, P., Ameloot, P., Van Craenenbroeck, K., De Rycke, R., & Callewaert, N. (2010). The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins. MICROBIAL CELL FACTORIES, 9.
Vancouver
1.
Guerfal M, Ryckaert S, Jacobs P, Ameloot P, Van Craenenbroeck K, De Rycke R, et al. The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins. MICROBIAL CELL FACTORIES. 2010;9.
MLA
Guerfal, Mouna, Stefan Ryckaert, Pieter Jacobs, et al. “The HAC1 Gene from Pichia Pastoris: Characterization and Effect of Its Overexpression on the Production of Secreted, Surface Displayed and Membrane Proteins.” MICROBIAL CELL FACTORIES 9 (2010): n. pag. Print.