Dataset Information

Decreased secretion and unfolded protein response up-regulation are correlated with intracellular retention for single-chain antibody variants produced in yeast.

ABSTRACT: Heterologous protein expression can easily overwhelm a cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. In the current model of the UPR, the chaperone BiP modulates the activation of the UPR due to its interactions with the signaling protein Ire1p and newly synthesized proteins. In this research, 4-4-20 scFv variants were generated by rational design to alter BiP binding to newly synthesized scFv proteins or via directed evolution aimed at improved secretion. Interestingly, the predicted BiP binding ability did not correlate significantly with the UPR. However, pulse-chase analysis of scFv fate revealed that mutants with a decreased ER residence time were more highly secreted, indicating that improved protein folding was more likely the cause for improved secretion. In fact, decreased secretion correlated with increased binding by BiP, as determined by co-immune precipitation studies. This suggests that the algorithm is not useful for in vivo prediction of variants, and that in vivo screens are more effective for finding variants with improved properties.

SUBMITTER: Xu P

PROVIDER: S-EPMC2744959 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Decreased secretion and unfolded protein response up-regulation are correlated with intracellular retention for single-chain antibody variants produced in yeast.

Xu Ping P Robinson Anne Skaja AS

Biotechnology and bioengineering 20090901 1

Heterologous protein expression can easily overwhelm a cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. In the current model of the UPR, the chaperone BiP modulates the activation of the UPR due to its interactions with the signaling protein Ire1p and newly synthesized proteins. In this research, 4-4-20 scFv variants were generated by rational design to alter BiP binding to newly synthesized scFv proteins or ...[more]

PMID: 19415776

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Project description:Ethanol is a valuable industrial product and a common metabolite used by many cell types. However, this molecule produces high levels of cytotoxicity affecting cellular performance at several levels. In the presence of ethanol, cells must adjust some of their components, such as the membrane lipids to maintain homeostasis. In the case of microorganism as Saccharomyces cerevisiae, ethanol is one of the principal products of their metabolism and is the main stress factor during fermentation. Although, many efforts have been made, mechanisms of ethanol tolerance are not fully understood and very little evidence is available to date for specific signaling by ethanol in the cell. This work studied two S. cerevisiae strains, CECT10094, and Temohaya-MI26, isolated from flor wine and agave fermentation (a traditional fermentation from Mexico) respectively, which differ in ethanol tolerance, in order to understand the molecular mechanisms underlying the ethanol stress response and the reasons for different ethanol tolerance. The transcriptome was analyzed after ethanol stress and, among others, an increased activation of genes related with the unfolded protein response (UPR) and its transcription factor, Hac1p, was observed in the tolerant strain CECT10094. We observed that this strain also resist more UPR agents than Temohaya-MI26 and the UPR-ethanol stress correlation was corroborated observing growth of 15 more strains and discarding UPR correlation with other stresses as thermal or oxidative stress. Furthermore, higher activation of UPR pathway in the tolerant strain CECT10094 was observed using a UPR mCherry reporter. Finally, we observed UPR activation in response to ethanol stress in other S. cerevisiae ethanol tolerant strains as the wine strains T73 and EC1118. This work demonstrates that the UPR pathway is activated under ethanol stress occurring in a standard fermentation and links this response to an enhanced ethanol tolerance. Thus, our data suggest that there is a room for ethanol tolerance improvement by enhancing UPR response.

Dataset Information

Decreased secretion and unfolded protein response up-regulation are correlated with intracellular retention for single-chain antibody variants produced in yeast.

Publications

Decreased secretion and unfolded protein response up-regulation are correlated with intracellular retention for single-chain antibody variants produced in yeast.

Similar Datasets

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