Person:
Jacobs, Pieter P.

Profile Picture

Email Address

AA Acceptance Date

Birth Date

Research Projects

Organizational Units

Job Title

Last Name

Jacobs

First Name

Pieter P.

Name

Jacobs, Pieter P.

Filters

20102

Settings

Author's Publications: search.filters.isAuthorOfPublication.a7325b14-4c5b-4585-a7cf-d98db8f8d4ee

Search Results

Now showing 1 - 2 of 2
  • Thumbnail Image
    Publication
    Fed-Batch Fermentation of GM-CSF-Producing Glycoengineered Pichia pastoris Under Controlled Specific Growth Rate
    (BioMed Central, 2010) Jacobs, Pieter P.; Inan, Mehmet; Festjens, Nele; Haustraete, Jurgen; Van Hecke, Annelies; Contreras, Roland; Meagher, Michael M; Callewaert, Nico
    Background: Yeast expression systems with altered N-glycosylation are now available to produce glycoproteins with homogenous, defined N-glycans. However, data on the behaviour of these strains in high cell density cultivation are scarce. Results: Here, we report on cultivations under controlled specific growth rate of a GlycoSwitch-Man5 Pichia pastoris strain producing Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) at high levels (hundreds of milligrams per liter). We demonstrate that homogenous Man5GlcNAc2 N-glycosylation of the secreted proteins is achieved at all specific growth rates tested. Conclusions: Together, these data illustrate that the GlycoSwitch-Man5 P. pastoris is a robust production strain for homogenously N-glycosylated proteins.
  • Thumbnail Image
    Publication
    The HAC1 Gene from Pichia Pastoris: Characterization and Effect of its Overexpression on the Production of Secreted, Surface Displayed and Membrane Proteins
    (BioMed Central, 2010) Guerfal, Mouna; Ryckaert, Stefan; Jacobs, Pieter P.; Ameloot, Paul; Van Craenenbroeck, Kathleen; Derycke, Riet; Callewaert, Nico
    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.