Abstract
One of the main limitations for heterologous protein production in the yeast Saccharomyces cerevisiae is the protein-folding capacity in the endoplasmic reticulum (ER). Accumulation of unfolded proteins triggers the unfolded protein response (UPR), which resolves the stress by increasing the capacity for protein folding and removal of unfolded proteins by the ER-associated degradation (ERAD) system. In order to analyze the influence of ERAD on production of a human IgG, we disrupted ERAD at different stages by deletion of the HTM1, YOS9, HRD1, HRD3, or UBC7 gene, with or without a disruption of the UPR by deletion of the IRE1 gene. All deletion strains were viable and did not exhibit a growth phenotype under normal growth conditions. Deletion of HTM1 resulted in a small increase in antibody production, whereas a small decrease in antibody production was observed in the Δhrd1, Δhrd3, and Δubc7 yeast strains, and a stronger decrease in the Δyos9 yeast strain. Deletion of the IRE1 gene had contrasting effects in the ERAD mutants, with a strongly decreased production in wild-type cells and partially reversed effects in combination with the Δhtm1 or the Δyos9 deletions. In order to study IgG clearance from the ER, an assay was developed using the inhibitory effect of glucose on the GAL1 promoter that is driving IgG expression. The Δyos9Δire1and Δhtm1Δire1 strains showed a delayed IgG clearance from the cells, showing that removal of components for the generation and recognition of the glycan signal needed for ERAD-mediated protein degradation might increase the IgG ER residence time.
Similar content being viewed by others
References
Apodaca J, Kim I, Rao H (2006) Cellular tolerance of prion protein PrP in yeast involves proteolysis and the unfolded protein response. Biochem Biophys Res Commun 347:319–326. doi:10.1016/j.bbrc.2006.06.078
Bailey U-M, Schulz BL (2013) Deglycosylation systematically improves N-glycoprotein identification in liquid chromatography-tandem mass spectrometry proteomics for analysis of cell wall stress responses in Saccharomyces cerevisiae lacking Alg3p. J Chromatogr B Anal Technol Biomed Life Sci 923–924:16–21. doi:10.1016/j.jchromb.2013.01.026
Benitez EM, Stolz A, Wolf DH (2011) Yos9, a control protein for misfolded glycosylated and non-glycosylated proteins in ERAD. FEBS Lett 585:3015–3019. doi:10.1016/j.febslet.2011.08.021
Carvalho NDSP, Arentshorst M, Kooistra R, Stam H, Sagt CM, van den Hondel CAMJJ, Ram AFJ (2011) Effects of a defective ERAD pathway on growth and heterologous protein production in Aspergillus niger. Appl Microbiol Biotechnol 89:357–373. doi:10.1007/s00253-010-2916-5
Clerc S, Hirsch C, Oggier DM, Deprez P, Jakob C, Sommer T, Aebi M (2009) Htm1 protein generates the N-glycan signal for glycoprotein degradation in the endoplasmic reticulum. J Cell Biol 184:159–172. doi:10.1083/jcb.200809198
Delic M, Göngrich R, Mattanovich D, Gasser B (2014) Engineering of protein folding and secretion-strategies to overcome bottlenecks for efficient production of recombinant proteins. Antioxid Redox Signal 21:414–437. doi:10.1089/ars.2014.5844
Friedlander R, Jarosch E, Urban J, Volkwein C, Sommer T (2000) A regulatory link between ER-associated protein degradation and the unfolded-protein response. Nat Cell Biol 2:379–384. doi:10.1038/35017001
Gasser B, Mattanovich D (2007) Antibody production with yeasts and filamentous fungi: on the road to large scale? Biotechnol Lett 29:201–212. doi:10.1007/s10529-006-9237-x
Gauss R, Kanehara K, Carvalho P, Ng DTW, Aebi M (2011) A complex of Pdi1p and the mannosidase Htm1p initiates clearance of unfolded glycoproteins from the endoplasmic reticulum. Mol Cell 42:782–793. doi:10.1016/j.molcel.2011.04.027
Hegemann JH, Heick SB (2011) Delete and repeat: a comprehensive toolkit for sequential gene knockout in the budding yeast Saccharomyces cerevisiae. Methods Mol Biol 765:189–206. doi:10.1007/978-1-61779-197-0
Helenius A, Aebi M (2004) Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73:1019–1049. doi:10.1146/annurev.biochem.73.011303.073752
Herscovics A (2001) Structure and function of Class I α1,2-mannosidases involved in glycoprotein synthesis and endoplasmic reticulum quality control. Biochimie 83:757–762
Hiller MM, Finger A, Schweiger M, Wolf DH (1996) ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway. Science 273:1725–1728
Hou J, Tyo KEJ, Liu Z, Petranovic D, Nielsen J (2012) Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae. FEMS Yeast Res 12:491–510. doi:10.1111/j.1567-1364.2012.00810.x
Izawa T, Nagai H, Endo T, Nishikawa S (2012) Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER-associated degradation. Mol Biol Cell 23:1283–1293. doi:10.1091/mbc.E11-08-0722
Jakob CA, Burda P, Roth J, Aebi M (1998) Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure. J Cell Biol 142:1223–1233
Li H, Sethuraman N, Stadheim TA, Zha D, Prinz B, Ballew N, Bobrowicz P, Choi B-K, Cook WJ, Cukan M, Houston-Cummings NR, Davidson R, Gong B, Hamilton SR, Hoopes JP, Jiang Y, Kim N, Mansfield R, Nett JH, Rios S, Strawbridge R, Wildt S, Gerngross TU (2006) Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat Biotechnol 24:210–215. doi:10.1038/nbt1178
Li S, Spooner RA, Hampton RY, Lord JM, Roberts LM (2012) Cytosolic entry of Shiga-like toxin a chain from the yeast endoplasmic reticulum requires catalytically active Hrd1p. PLoS ONE 7:e41119. doi:10.1371/journal.pone.0041119
Mumberg D, Müller R, Funk M (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156:119–122
Nakatsukasa K, Brodsky JL, Kamura T (2013) A stalled retrotranslocation complex reveals physical linkage between substrate recognition and proteasomal degradation during ER-associated degradation. Mol Biol Cell 24:1765–1775. doi:10.1091/mbc.E12-12-0907
Parsaie Nasab F, Aebi M, Bernhard G, Frey AD (2013) A combined system for engineering glycosylation efficiency and glycan structure in Saccharomyces cerevisiae. Appl Environ Microbiol 79:997–1007. doi:10.1128/AEM.02817-12
Pfeffer M, Maurer M, Stadlmann J, Grass J, Delic M, Altmann F, Mattanovich D (2012) Intracellular interactome of secreted antibody Fab fragment in Pichia pastoris reveals its routes of secretion and degradation. Appl Microbiol Biotechnol 93:2503–2512. doi:10.1007/s00253-012-3933-3
Piirainen MA, de Ruijter JC, Koskela EV, Frey AD (2014) Glycoengineering of yeasts from the perspective of glycosylation efficiency. N Biotechnol 31:532–537. doi:10.1016/j.nbt.2014.03.001
Potgieter TI, Cukan M, Drummond JE, Houston-Cummings NR, Jiang Y, Li F, Lynaugh H, Mallem M, McKelvey TW, Mitchell T, Nylen A, Rittenhour A, Stadheim TA, Zha D, d’Anjou M (2009) Production of monoclonal antibodies by glycoengineered Pichia pastoris. J Biotechnol 139:318–325. doi:10.1016/j.jbiotec.2008.12.015
Potgieter TI, Kersey SD, Mallem MR, Nylen AC, d’Anjou M (2010) Antibody expression kinetics in glycoengineered Pichia pastoris. Biotechnol Bioeng 106:918–927. doi:10.1002/bit.22756
Quan EM, Kamiya Y, Kamiya D, Denic V, Weibezahn J, Kato K, Weissman JS (2008) Defining the glycan destruction signal for endoplasmic reticulum-associated degradation. Mol Cell 32:870–877. doi:10.1016/j.molcel.2008.11.017
Szathmary R, Bielmann R, Nita-Lazar M, Burda P, Jakob CA (2005) Yos9 protein is essential for degradation of misfolded glycoproteins and may function as lectin in ERAD. Mol Cell 19:765–775. doi:10.1016/j.molcel.2005.08.015
Taxis C, Knop M (2006) System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae. BioTechniques 40:73–78. doi:10.2144/000112040
Valkonen M, Penttilä M, Saloheimo M (2003) Effects of inactivation and constitutive expression of the unfolded- protein response pathway on protein production in the yeast Saccharomyces cerevisiae. Appl Environ Microbiol 69:2065–2072
Wittrup KD, Robinson AS, Parekh RN, Forrester KJ (1994) Existence of an optimum expression level for secretion of foreign proteins in yeast. Ann N Y Acad Sci 745:321–330
Acknowledgments
The work was supported by Aalto University Bioeconomy facilities. We would like to acknowledge the support of Dr. Mari Valkonen (VTT, Finland) for assistance with the imaging system.
Conflict of interest
The authors declare that they have no competing interests.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
de Ruijter, J.C., Frey, A.D. Analysis of antibody production in Saccharomyces cerevisiae: effects of ER protein quality control disruption. Appl Microbiol Biotechnol 99, 9061–9071 (2015). https://doi.org/10.1007/s00253-015-6807-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-015-6807-7