Dataset Information

Low Shear Stress Increases Recombinant Protein Production and High Shear Stress Increases Apoptosis in Human Cells.

ABSTRACT: Human embryonic kidney cells HEK293 can be used for the production of therapeutic glycoproteins requiring human post-translational modifications. High cell density perfusion processes are advantageous for such production but are challenging due to the shear sensitivity of HEK293 cells. To understand the impact of hollow filter cell separation devices, cells were cultured in bioreactors operated with tangential flow filtration (TFF) or alternating tangential flow filtration (ATF) at various flow rates. The average theoretical velocity profile in these devices showed a lower shear stress for ATF by a factor 0.637 compared to TFF. This was experimentally validated and, furthermore, transcriptomic evaluation provided insights into the underlying cellular processes. High shear caused cellular stress leading to apoptosis by three pathways, i.e. endoplasmic reticulum stress, cytoskeleton reorganization, and extrinsic signaling pathways. Positive effects of mild shear stress were observed, with increased recombinant erythropoietin production and increased gene expression associated with transcription and protein phosphorylation.

SUBMITTER: Zhan C

PROVIDER: S-EPMC7593556 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Low Shear Stress Increases Recombinant Protein Production and High Shear Stress Increases Apoptosis in Human Cells.

Zhan Caijuan C Bidkhori Gholamreza G Schwarz Hubert H Malm Magdalena M Mebrahtu Aman A Field Ray R Sellick Christopher C Hatton Diane D Varley Paul P Mardinoglu Adil A Rockberg Johan J Chotteau Veronique V

iScience 20201007 11

Human embryonic kidney cells HEK293 can be used for the production of therapeutic glycoproteins requiring human post-translational modifications. High cell density perfusion processes are advantageous for such production but are challenging due to the shear sensitivity of HEK293 cells. To understand the impact of hollow filter cell separation devices, cells were cultured in bioreactors operated with tangential flow filtration (TFF) or alternating tangential flow filtration (ATF) at various flow ...[more]

PMID: 33145483

Similar Datasets

Project description:In Escherichia coli, many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is-besides the produced target-the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in E. coliIMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm of E. coli, we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.

Dataset Information

Low Shear Stress Increases Recombinant Protein Production and High Shear Stress Increases Apoptosis in Human Cells.

Publications

Low Shear Stress Increases Recombinant Protein Production and High Shear Stress Increases Apoptosis in Human Cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets