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Optimizing Recombinant Protein Production in the Escherichia coli Periplasm Alleviates Stress
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Number of Authors: 42018 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 84, no 12, article id e00270Article in journal (Refereed) Published
Abstract [en]

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. coli.

IMPORTANCE 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.

Place, publisher, year, edition, pages
2018. Vol. 84, no 12, article id e00270
Keywords [en]
Escherichia coli, recombinant protein production, periplasm, Sec translocon, proteomics
National Category
Environmental Biotechnology Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-157686DOI: 10.1128/AEM.00270-18ISI: 000433902200007PubMedID: 29654183OAI: oai:DiVA.org:su-157686DiVA, id: diva2:1236061
Available from: 2018-07-31 Created: 2018-07-31 Last updated: 2018-08-17Bibliographically approved
In thesis
1. Protein production in the E. coli cell envelope
Open this publication in new window or tab >>Protein production in the E. coli cell envelope
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins fulfil essential functions in every cell and malfunctioning proteins are often the cause of diseases. On the other hand, proteins like antibody fragments or hormones can be used to treat diseases. Proteins are often produced in the bacterium Escherichia coli so that they can be studied to understand their (mal)function or so that they can be used to treat a disease. Unfortunately, producing proteins in the cell envelope of E. coli, like integral membrane proteins, which are important drug targets, and secretory proteins like antibody fragments and hormones, often results in unsatisfactory yields. Therefore, the objectives of this doctoral thesis were to identify bottlenecks that can limit the production of recombinant proteins in the cell envelope of E. coli and to try to overcome these bottlenecks. In the first study, we isolated and characterized the E. coli membrane protein production strain Mt56(DE3). This strain, in which the target gene expression intensity is strongly reduced, outcompetes the standard E. coli membrane protein production strains for most targets tested. In the second and third study we focused on the production of secretory proteins, i.e., proteins that are translocated across the inner membrane into the periplasm of E. coli. First, we investigated the impact of the targeting pathway used to direct a secretory protein to the translocation machinery on the cell physiology and protein production yields. We found that the co-translational targeting of a produced protein saturates the capacity of the translocation machinery resulting in heavily impaired biomass formation and low protein production yields. In contrast, post-translational targeting of a produced protein did not saturate the capacity of the protein translocation machinery resulting in hardly affected biomass formation and high protein production yields. In the third study we investigated how optimizing the production of a co-translationally targeted protein, by harmonizing its production rate with the capacity of the protein translocation machinery, affects the physiology of the cell. We found that, in stark contrast to the non-optimized condition, the optimized production did not affect the composition of the E. coli proteome. This surprising finding indicates that a protein can be produced efficiently in the periplasm of E. coli without compromising the physiology of the cell. In the last study we aimed at developing an outer membrane vesicle-based tuberculosis vaccine. To this end, an E. coli strain was created that produced outer membrane vesicles coated with different tuberculosis antigens. It was shown that a homogenous population of vesicles was produced, which will hopefully facilitate the isolation of these vesicles on an industrial scale.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018. p. 91
Keywords
E. coli, protein biogenesis, recombinant proteins, membrane proteins, secretory proteins, protein displays, outer membrane vesicles
National Category
Biochemistry and Molecular Biology Microbiology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-158451 (URN)978-91-7797-402-4 (ISBN)978-91-7797-403-1 (ISBN)
Public defence
2018-10-08, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2018-09-13 Created: 2018-08-15 Last updated: 2018-09-04Bibliographically approved

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