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Sensing and analyzing unfolded protein response during heterologous protein production

2017-04-21 14:45:09 | 日記

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The recombinant proteins production is critical for biotechnology and biomedical research. Heterologous protein expression can saturate the cell’s capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. Our goals were to detect and analyze the UPR during heterologous protein expression, understand its mechanism and regulation, and develop strategies to reduce the stress response for the improvement of recombinant protein production.
UPR during the expression of the single-chain antibody 4-4-20 (scFv) in yeast Saccharomyces cerevisiae was explored in several ways. Overexpression of the chaperone BiP did not reduce the UPR activated by scFv expression; however, overexpression of the foldase PDI or co-overexpression of BiP and PDI did reduce the UPR. It was observed that co-overexpression of BiP and PDI led to the greatest secretion of scFv from the cell, but BiP and PDI appear to interact with the newly synthesized scFv at different stages in the folding process, as determined by pulse-chase analysis. BiP appears to act primarily to facilitate translocation and retain unfolded or partially-folded scFv, and PDI actively folds the scFv through its functions as a catalyst, and /or an isomerase, of disulfide bonds, relieving the unfolding stress of the cells.
For understanding the genomic UPR regulation during scFv production, cDNA microarray analysis was employed and the gene regulation was compared to the UPR induced by chemical treatment. Analysis of microarray data using a novel probabilistic framework, which enabled us to identify UPR target genes with a much Documents PDF Complete Click Here & Upgrade Expanded Features Unlimited Pages PREVIEW xvii greater enrichment than that using a two-fold change approach, reveals that a significant number of UPR target genes were up-regulated during 4-4-20 scFv expression, showing that the UPR activated by scFv expression has a wide scope of regulation, which includes protein folding, protein degradation, and protein secretion. The study also shows that the different unfolded protein response elements (UPRE-1, UPRE-2 and UPRE-3) could confer the unfolded protein response on the target genes during scFv expression. The experimental and statistical analyses indicate that the unfolded protein response activated by 4-4-20 scFv expression closely resembles that induced by chemical treatment.
In order to get a more thorough understanding of the roles of BiP in the UPR, the effect of chaperone BiP binding to unfolded proteins was investigated using different 4-4-20 scFv variants, which were obtained from rational design or directed evolution. The study shows that the unfolded protein response was not only affected by the binding ability of BiP to unfolded proteins, but also likely affected by the scFv folding properties themselves. The decrease in the ability of protein binding to BiP did not always lead to a decreased unfolded protein response; however, an improvement in protein folding did decrease the unfolded protein response and improve protein secretion. These comprehensive UPR studies during 4-4-20 scFv expression by different molecular approaches is valuable for understanding the mechanism of UPR activation and improving protein production via cellular or protein engineering.
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