The HAC1 Gene from Pichia Pastoris: Characterization and Effect of its Overexpression on the Production of Secreted, Surface Displayed and Membrane Proteins

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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

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dc.contributor.author Guerfal, Mouna
dc.contributor.author Ryckaert, Stefan
dc.contributor.author Ameloot, Paul
dc.contributor.author Van Craenenbroeck, Kathleen
dc.contributor.author Derycke, Riet
dc.contributor.author Callewaert, Nico
dc.contributor.author Jacobs, Pieter P.
dc.date.accessioned 2011-04-18T00:57:02Z
dc.date.issued 2010
dc.identifier.citation Guerfal, Mouna, Stefan Ryckaert, Pieter P. Jacobs, Paul Ameloot, Kathleen Van Craenenbroeck, Riet Derycke, 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: 49. en_US
dc.identifier.issn 1475-2859 en_US
dc.identifier.uri http://nrs.harvard.edu/urn-3:HUL.InstRepos:4853399
dc.description.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, α-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. en_US
dc.language.iso en_US en_US
dc.publisher BioMed Central en_US
dc.relation.isversionof doi:10.1186/1475-2859-9-49 en_US
dc.relation.hasversion http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2905327/pdf/ en_US
dash.license LAA
dc.title The HAC1 Gene from Pichia Pastoris: Characterization and Effect of its Overexpression on the Production of Secreted, Surface Displayed and Membrane Proteins en_US
dc.type Journal Article en_US
dc.description.version Version of Record en_US
dc.relation.journal Microbial Cell Factories en_US
dash.depositing.author Jacobs, Pieter P.
dc.date.available 2011-04-18T00:57:02Z
dash.affiliation.other HMS^Dermatology-Brigham and Women's Hospital en_US

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