caa a22 4500 605505810 CHVBK 20210128100623.0 cr unu---uuuuu 210128e20151101xx s 000 0 eng 10.1007/s00253-015-6807-7 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6807-7 Analysis of antibody production in Saccharomyces cerevisiae [Elektronische Daten] effects of ER protein quality control disruption [Jorg de Ruijter, Alexander Frey] 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. Springer-Verlag Berlin Heidelberg, 2015 Unfolded protein response (UPR) nationallicence Endoplasmic reticulum associated protein degradation (ERAD) nationallicence Heterologous protein production nationallicence Yeast nationallicence Antibody nationallicence de Ruijter Jorg Department of Biotechnology and Chemical Technology, Aalto University, Kemistintie 1, 02150, Espoo, Finland aut Frey Alexander Department of Biotechnology and Chemical Technology, Aalto University, Kemistintie 1, 02150, Espoo, Finland aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/21(2015-11-01), 9061-9071 0175-7598 99:21<9061 2015 99 253 https://doi.org/10.1007/s00253-015-6807-7 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6807-7 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- de Ruijter Jorg Department of Biotechnology and Chemical Technology, Aalto University, Kemistintie 1, 02150, Espoo, Finland aut NATIONALLICENCE 700 1- Frey Alexander Department of Biotechnology and Chemical Technology, Aalto University, Kemistintie 1, 02150, Espoo, Finland aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/21(2015-11-01), 9061-9071 0175-7598 99:21<9061 2015 99 253