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Protein production in the E. coli cell envelope
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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 [en]
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: urn:nbn:se:su:diva-158451ISBN: 978-91-7797-402-4 (print)ISBN: 978-91-7797-403-1 (electronic)OAI: oai:DiVA.org:su-158451DiVA, id: diva2:1238939
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
List of papers
1. Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)
Open this publication in new window or tab >>Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 45089Article in journal (Refereed) Published
Abstract [en]

Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived from the T7 RNA polymerase (P)-based BL21(DE3) protein production strain. In C41(DE3) and C43(DE3) mutations lowering t7rnap expression levels result in strongly reduced T7 RNAP accumulation levels. As a consequence membrane protein production stress is alleviated in the C41(DE3) and C43(DE3) strains, thereby increasing membrane protein yields. Here, we isolated Mutant56(DE3) from BL21(DE3) using a genetic screen designed to isolate BL21(DE3)-derived strains with mutations alleviating membrane protein production stress other than the ones in C41(DE3) and C43(DE3). The defining mutation of Mutant56(DE3) changes one amino acid in its T7 RNAP, which weakens the binding of the T7 RNAP to the T7 promoter governing target gene expression rather than lowering T7 RNAP levels. For most membrane proteins tested yields in Mutant56(DE3) were considerably higher than in C41(DE3) and C43(DE3). Thus, the isolation of Mutant56(DE3) shows that the evolution of BL21(DE3) can be promoted towards further enhanced membrane protein production.

National Category
Biological Sciences Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-142640 (URN)10.1038/srep45089 (DOI)000397309700001 ()
Available from: 2017-05-15 Created: 2017-05-15 Last updated: 2018-08-17Bibliographically approved
2. Post-translational targeting of a recombinant protein promotes its efficient secretion into the E. coli periplasm
Open this publication in new window or tab >>Post-translational targeting of a recombinant protein promotes its efficient secretion into the E. coli periplasm
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-158450 (URN)
Available from: 2018-08-03 Created: 2018-08-03 Last updated: 2018-08-15Bibliographically approved
3. Optimizing Recombinant Protein Production in the Escherichia coli Periplasm Alleviates Stress
Open this publication in new window or tab >>Optimizing Recombinant Protein Production in the Escherichia coli Periplasm Alleviates Stress
2018 (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.

Keywords
Escherichia coli, recombinant protein production, periplasm, Sec translocon, proteomics
National Category
Environmental Biotechnology Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-157686 (URN)10.1128/AEM.00270-18 (DOI)000433902200007 ()29654183 (PubMedID)
Available from: 2018-07-31 Created: 2018-07-31 Last updated: 2018-08-17Bibliographically approved
4. Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach
Open this publication in new window or tab >>Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach
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2014 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 18, p. 5854-5865Article in journal (Refereed) Published
Abstract [en]

Outer membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of the Mycobacterium tuberculosis antigens ESAT6, Ag85B, and Rv2660c were targeted to the surface of Escherichia coli OMVs upon fusion to Hbp. Furthermore, a hypervesiculating Delta tolR Delta tolA derivative of attenuated Salmonella enterica serovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by the M. tuberculosis antigens and epitopes from Chlamydia trachomatis major outer membrane protein (MOMP). Also, we showed that delivery of Salmonella OMVs displaying Ag85B to antigen-presenting cells in vitro results in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-107981 (URN)10.1128/AEM.01941-14 (DOI)000341488200032 ()
Note

AuthorCount:19;

Available from: 2014-10-09 Created: 2014-10-06 Last updated: 2018-08-17Bibliographically approved

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