Analysis of antibody production in Saccharomyces cerevisiae: effects of ER protein quality control disruption

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 analyse 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.3

Enhancing antibody folding and secretion by tailoring the Saccharomyces cerevisiae endoplasmic reticulum

“…When using cellular engineering to improve heterologous protein production, it is also useful to gain understanding of the underlying changes in strains with interesting secretion phenotypes. To gain more insight in how some of the overexpressed folding factors influence the progression of the antibody through the cell, we selected five strains for use in a cellular clearance assay as described in De Ruijter and Frey [ 12 ]. The strains were as follows: the two genetic strain backgrounds; wt and Δopi1, the highest producers from both backgrounds; wt with the CPR5 -P KAR2 plasmid, and Δopi1 with the CPR5 -P GPD plasmid, and finally one of the strains with a negatively affecting gene combination: Δopi1 with the KAR2 -P GAL1 and the LHS1 -P GAL1 plasmids.…”

“…The plasmid for the IgG gene integration was achieved as follows: a fragment from pEK5 plasmid [ 12 ], containing genes for heavy and light chain each under GAL1-promoter, was amplified with PCR by using the oligonucleotides 1 and 2. These oligonucleotides contained complementary 5′ ends to the pRS303N-plasmid [ 66 ].…”

“…The UPR is a transcriptional program for concomitant upregulation of over 300 genes, including the upregulation of basal expression levels of constitutively expressed quality control components in the ER, which include folding factors, chaperones and the ER associated degradation (ERAD) machinery [ 8 – 10 ]. Inactivation of the UPR has shown to be detrimental for heterologous protein production [ 11 , 12 ]. Although the UPR response is shown to be specific to the source of stress [ 13 ], heterologous protein yields could benefit most from a protein specific, fine-tuned expression of necessary helper proteins.…”

Filamentous fungi-like secretory pathway strayed in a yeast system: peculiarities of Yarrowia lipolytica secretory pathway underlying its extraordinary performance

“…The authors concluded that excessive protein production could negatively affect protein folding due to deficiency in chaperones and saturation of the secretion machinery (Dulermo et al 2017). It has been postulated that folding, maturation, and intracellular transportation constitute the main bottlenecks of the overall process of recombinant protein production (Gasser et al 2007a; de Ruijter and Frey 2015; Puxbaum et al 2015; Tang et al 2015). Depending on the specific properties of the targeted polypeptide, i.e., primary and secondary structure, oligomerization, glycosylation, and the ultimate destination, different phenomena may limit the overall efficiency of the secretory protein synthesis.…”

Improved recombinant protein production in Arabidopsis thaliana

“…trigger the unfolded protein response/ER-associated degradation). [40][41][42][43][44] When a protein of interest should not accumulate to satisfactorily high levels, one could apply various genetic manipulations, including chemical mutagenesis, activation tagging, 45 and targeted/untargeted overexpression (for example, FOX hunting) 46 to the transgenic line of interest and screen for individuals with high expression levels, like used in bacterial engineering. 47 The factors identified by such approaches can also be incorporated into other large-scale plant expression systems, via CRISPR/Cas9-targeted mutagenesis or overexpression of endogenous factors.…”