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Enhancing Recombinant Protein Yields in the E. coli Periplasm by Combining Signal Peptide and Production Rate Screening
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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Number of Authors: 92019 (English)In: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 10, article id 1511Article in journal (Refereed) Published
Abstract [en]

Proteins that contain disulfide bonds mainly mature in the oxidative environment of the eukaryotic endoplasmic reticulum or the periplasm of Gram-negative bacteria. In E. coli, disulfide bond containing recombinant proteins are often targeted to the periplasm by an N -terminal signal peptide that is removed once it passes through the Sectranslocon in the cytoplasmic membrane. Despite their conserved targeting function, signal peptides can impact recombinant protein production yields in the periplasm, as can the production rate. Here, we present a combined screen involving different signal peptides and varying production rates that enabled the identification of more optimal conditions for periplasmic production of recombinant proteins with disulfide bonds. The data was generated from two targets, a single chain antibody fragment (BL1) and human growth hormone (hGH), with four different signal peptides and a titratable rhamnose promoter-based system that enables the tuning of protein production rates. Across the screen conditions, the yields for both targets significantly varied, and the optimal signal peptide and rhamnose concentration differed for each protein. Under the optimal conditions, the periplasmic BL1 and hGH were properly folded and active. Our study underpins the importance of combinatorial screening approaches for addressing the requirements associated with the production of a recombinant protein in the periplasm.

Place, publisher, year, edition, pages
2019. Vol. 10, article id 1511
Keywords [en]
Escherichia coil, recombinant protein, periplasm, signal peptide, protein production rate, protein production screen
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-171632DOI: 10.3389/fmicb.2019.01511ISI: 000476731900001PubMedID: 31396164OAI: oai:DiVA.org:su-171632DiVA, id: diva2:1345034
Available from: 2019-08-22 Created: 2019-08-22 Last updated: 2024-01-17Bibliographically approved
In thesis
1. Membrane and secretory protein production in Escherichia coli
Open this publication in new window or tab >>Membrane and secretory protein production in Escherichia coli
2021 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Escherichia coli (E. coli) is the most widely used bacterium for the production of recombinant proteins. However, the production of proteins in its cytoplasmic membrane or periplasm is challenging. Therefore, the aim of this doctoral thesis was to develop setups for enhancing the production of membrane and secretory proteins in E. coli. Production of membrane and secretory proteins in E. coli requires precise tuning of protein production rates in order to avoid the deleterious saturation of the secretion apparatus. Here, we engineered two tunable protein production setups. We engineered the pReX expression vector, which is a simplified version of the setup used for creating the T7 RNA polymerase-based Lemo21(DE3) strain, and a setup based on the use of the rhamnose promoter in a Δrha strain background. Both setups can be used to enhance membrane and secretory protein production yields. Based on our current knowledge, it is challenging to predict which signal peptide should be utilized to produce a recombinant protein in the periplasm. Using the tunable rhamnose promoter-based setup, we developed a combined screen involving different signal peptides and varying production rates. This enables the identification of an optimal signal peptide and production rate combination for the periplasmic production of a recombinant protein. Next, proteome analysis was used to examine cells producing a recombinant protein in the periplasm, when using, an optimal signal peptide ­and protein production rate combination. Interestingly, the proteome analysis showed that cells had increased their protein translocation capacity, i.e., they had adapted. Finally, we investigated the co-translational folding of the periplasmic, disulfide bond-containing, E. coli protein alkaline phosphatase. Using Force Profile Analysis, it was shown that co-translationally translocated PhoA folds via at least two disulfide-stabilized folding intermediates. 

This thesis highlights the importance of fine-tuning membrane and secretory protein production rates to enhance their production yields, selecting the most optimal signal peptide for the periplasmic production of a protein, using combinatorial protein production screening approaches, studying the effects of recombinant protein production on the cell and developing experimental systems to monitor periplasmic protein folding.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2021. p. 77
Keywords
Escherichia coli, recombinant protein, tunable protein production, cytoplasmic membrane, periplasm, signal peptide, force profile analysis, co-translational folding
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-197675 (URN)978-91-7911-648-4 (ISBN)978-91-7911-649-1 (ISBN)
Public defence
2021-12-01, online via Zoom, public link is available at the department website, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2021-11-08 Created: 2021-10-13 Last updated: 2022-02-25Bibliographically approved

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Karyolaimos, AlexandrosAmpah-Korsah, HenryDaniels, Robertde Gier, Jan-Willem

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