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Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins.

ABSTRACT: Lipopolysaccharide (LPS), also referred to as endotoxin, is the major constituent of the outer leaflet of the outer membrane of virtually all Gram-negative bacteria. The lipid A moiety, which anchors the LPS molecule to the outer membrane, acts as a potent agonist for Toll-like receptor 4/myeloid differentiation factor 2-mediated pro-inflammatory activity in mammals and, thus, represents the endotoxic principle of LPS. Recombinant proteins, commonly manufactured in Escherichia coli, are generally contaminated with endotoxin. Removal of bacterial endotoxin from recombinant therapeutic proteins is a challenging and expensive process that has been necessary to ensure the safety of the final product.As an alternative strategy for common endotoxin removal methods, we have developed a series of E. coli strains that are able to grow and express recombinant proteins with the endotoxin precursor lipid IVA as the only LPS-related molecule in their outer membranes. Lipid IVA does not trigger an endotoxic response in humans typical of bacterial LPS chemotypes. Hence the engineered cells themselves, and the purified proteins expressed within these cells display extremely low endotoxin levels.This paper describes the preparation and characterization of endotoxin-free E. coli strains, and demonstrates the direct production of recombinant proteins with negligible endotoxin contamination.

SUBMITTER: Mamat U

PROVIDER: S-EPMC4404585 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins.

Mamat Uwe U Wilke Kathleen K Bramhill David D Schromm Andra Beate AB Lindner Buko B Kohl Thomas Andreas TA Corchero José Luis JL Villaverde Antonio A Schaffer Lana L Head Steven Robert SR Souvignier Chad C Meredith Timothy Charles TC Woodard Ronald Wesley RW

Microbial cell factories 20150416

<h4>Background</h4>Lipopolysaccharide (LPS), also referred to as endotoxin, is the major constituent of the outer leaflet of the outer membrane of virtually all Gram-negative bacteria. The lipid A moiety, which anchors the LPS molecule to the outer membrane, acts as a potent agonist for Toll-like receptor 4/myeloid differentiation factor 2-mediated pro-inflammatory activity in mammals and, thus, represents the endotoxic principle of LPS. Recombinant proteins, commonly manufactured in Escherichia ...[more]

PMID: 25890161

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Project description:Modeling and analysis of genetic networks have become increasingly important in the investigation of cellular processes. The genetic networks involved in cellular stress response can have a critical effect on the productivity of recombinant proteins. In this work, it was found that the temperature-inducible expression system for the production of soluble recombinant streptokinase in Escherichia coli resulted in a lower productivity compared to the chemically-induced system. To investigate the effect of the induced cellular response due to temperature up-shift a model-based approach is adopted. The role played by the major molecular chaperone teams DnaK-DnaJ-GrpE and GroEL-GroES on the productivity of recombinant streptokinase was experimentally determined. Based on these investigations, a detailed mechanistic mathematical model was developed for the cellular response during the temperature-induced recombinant streptokinase production. The model simulations were found to have a good qualitative agreement with the experimental results. The mechanistic mathematical model was validated with the experiments conducted on a sigma(32) mutant strain. Detailed analysis of the parameter sensitivities of the model indicated that the level of free DnaK chaperone in the cell has the major effect on the productivity of recombinant streptokinase during temperature induction. Analysis of the model simulations also shows that down regulation or selective redirection of the heat shock proteins could be a better way of manipulating the cellular stress response than overexpression or deletion. In other words, manipulating the system properties resulting from the interaction of the components is better than manipulating the individual components. Although our results are specific to a recombinant protein (streptokinase) and the expression system (E. coli), we believe that such a systems-biological approach has several advantages over conventional experimental approaches and could be in principle extended to bigger genetic networks as well as other recombinant proteins and expression systems.

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

Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins.

Publications

Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins.

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