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Post-translational targeting of a recombinant protein promotes its efficient secretion into the E. coli periplasm
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-158450OAI: oai:DiVA.org:su-158450DiVA, id: diva2:1236581
Tillgänglig från: 2018-08-03 Skapad: 2018-08-03 Senast uppdaterad: 2018-08-15Bibliografiskt granskad
Ingår i avhandling
1. Protein production in the E. coli cell envelope
Öppna denna publikation i ny flik eller fönster >>Protein production in the E. coli cell envelope
2018 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018. s. 91
Nyckelord
E. coli, protein biogenesis, recombinant proteins, membrane proteins, secretory proteins, protein displays, outer membrane vesicles
Nationell ämneskategori
Biokemi och molekylärbiologi Mikrobiologi
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:su:diva-158451 (URN)978-91-7797-402-4 (ISBN)978-91-7797-403-1 (ISBN)
Disputation
2018-10-08, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (Engelska)
Opponent
Handledare
Anmärkning

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Tillgänglig från: 2018-09-13 Skapad: 2018-08-15 Senast uppdaterad: 2018-09-04Bibliografiskt granskad

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Av författaren/redaktören
Ytterberg, A. JimmyZubarev, Roman A.Baumgarten, Thomas
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Institutionen för biokemi och biofysik
Biokemi och molekylärbiologi

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