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Optimizing membrane and secretory protein production in Gram-negative bacteria
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins fulfil a wide variety of essential functions in the cell. Recombinant protein production in the Gram-negative bacterium Escherichia coli (E. coli) facilitates structural and functional studies of proteins and it has been instrumental in biotechnology for the manufacturing of e.g. many protein-based drugs. However, to obtain sufficient amounts of active recombinant protein is not always a trivial task. The production of proteins that reside in membranes is limited by their complex biogenesis and their hydrophobic nature. Consequently, despite the importance of membrane proteins in health and disease (approx. 70% of today’s drugs act on membrane proteins), structures of only a very small fraction of the existing membrane proteins have yet been solved. Many soluble proteins are also difficult to produce, e.g. those containing disulfide bonds (e.g. antibody fragments and most hormones). Disulfide bond-containing proteins have to be produced in the periplasm of E. coli to be able to fold properly. To reach the periplasm, these proteins have to be ‘secreted’ across the inner membrane, which makes that also their biogenesis is complex. The aim of my PhD studies has been to improve E. coli-based production of recombinant membrane and secretory proteins. I have found that (i) the previously developed Lemo21(DE3) protein production strain can be used to set the expression intensity of a gene encoding a membrane protein such that the protein is optimally produced in the cytoplasmic membrane without causing any notable stress. Also, (ii) membrane protein production using the Lemo21(DE3) strain can be improved and simplified using carefully optimized culturing and induction conditions, demonstrated by the development of the ‘MemStar recipe’. Furthermore, I found that (iii) when using the standard BL21(DE3)/pT7 expression system for the production of membrane and secretory proteins, omitting the inducer IPTG leads to drastically improved yields as compared to when IPTG is added, owing to a lower initial target protein production rate. In the fourth study, I found that (iv) when using the PrhaBAD promoter for expression of the target gene, protein accumulation rates appear to be mostly unaffected by the inducer concentration. Using a strain-engineering approach, PrhaBAD-based protein production rates could be made constant and rhamnose concentration dependent. This dramatically improved production yields of both membrane and secretory proteins, using only very low amounts of inducer. Taken together, in accordance with previous studies, lowering production rates is an efficient strategy to increase production yields of both membrane- and secretory proteins. This is mostly due to alleviating saturation of the machinery involved in the biogenesis of these proteins. Finally, I also conducted a study (v) where, in both E. coli and Salmonella, I orchestrated the production of two membrane proteins (one that mediates the production of antigens on the surface of Gram-negative bacteria and another that makes defined pore-structures in the Gram-negative bacterial cell envelope) for the development of a safe (non-living) vaccine platform.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2015.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-123418ISBN: 978-91-7649-284-0 (print)OAI: oai:DiVA.org:su-123418DiVA: diva2:873984
Public defence
2016-01-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

 

Available from: 2015-12-21 Created: 2015-11-25 Last updated: 2015-12-10Bibliographically approved
List of papers
1. Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3)
Open this publication in new window or tab >>Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3)
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2012 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 423, no 4, 648-659 p.Article in journal (Refereed) Published
Abstract [en]

Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The 17 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the 17 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concentrations of rhamnose indicated that this strain may be very suitable for optimizing the production of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the 17 RNAP by the 17 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by reduction of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies.

Keyword
membrane protein production, optimization of protein expression, membrane protein biogenesis, 17 RNA polymerase-based overexpression, membrane protein functional/structural studies
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-83039 (URN)10.1016/j.jmb.2012.07.019 (DOI)000310415800017 ()
Note

AuthorCount:9;

Available from: 2012-12-04 Created: 2012-12-03 Last updated: 2017-12-07Bibliographically approved
2. MemStar: A one-shot Escherichia coli-based approach for high-level bacterial membrane protein production
Open this publication in new window or tab >>MemStar: A one-shot Escherichia coli-based approach for high-level bacterial membrane protein production
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2014 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 588, no 20, 3761-3769 p.Article in journal (Refereed) Published
Abstract [en]

Optimising membrane protein production yields in Escherichia coli can be time- and resource-consuming. Here, we present a simple and effective Membrane protein Single shot amplification recipe: MemStar. This one-shot amplification recipe is based on the E. coli strain Lemo21(DE3), the PASM-5052 auto-induction medium and, contradictorily, an IPTG induction step. Using MemStar, production yields for most bacterial membrane proteins tested were improved to reach an average of 5 mg L-1 per OD600 unit, which is significantly higher than yields obtained with other common production strategies. With MemStar, we have been able to obtain new structural information for several transporters, including the sodium/proton antiporter NapA. (C) 2014 Federation of European Biochemical Societies.

Keyword
Membrane protein production, X-ray crystallography, High-throughput, Escherichia coli
National Category
Biochemistry and Molecular Biology Cell Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-108362 (URN)10.1016/j.febslet.2014.08.025 (DOI)000341993000015 ()
Note

AuthorCount:9;

Available from: 2014-10-24 Created: 2014-10-22 Last updated: 2017-12-05Bibliographically approved
3. High-level production of membrane proteins in E-coli BL21(DE3) by omitting the inducer IPTG
Open this publication in new window or tab >>High-level production of membrane proteins in E-coli BL21(DE3) by omitting the inducer IPTG
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2015 (English)In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, 142Article in journal (Refereed) Published
Abstract [en]

Background: For membrane protein production, the Escherichia coli T7 RNA polymerase (T7 RNAP)-based protein production strain BL21(DE3) in combination with T7-promoter based expression vectors is widely used. Cells are routinely cultured in Lysogeny broth (LB medium) and expression of the chromosomally localized t7rnap gene is governed by the isopropyl-beta-D-1-thiogalactopyranoside (IPTG) inducible lacUV5 promoter. The T7 RNAP drives the expression of the plasmid borne gene encoding the recombinant membrane protein. Production of membrane proteins in the cytoplasmic membrane rather than in inclusion bodies in a misfolded state is usually preferred, but often hampered due to saturation of the capacity of the Sec-translocon, resulting in low yields. Results: Contrary to expectation we observed that omission of IPTG from BL21(DE3) cells cultured in LB medium can lead to significantly higher membrane protein production yields than when IPTG is added. In the complete absence of IPTG cultures stably produce membrane proteins in the cytoplasmic membrane, whereas upon the addition of IPTG membrane proteins aggregate in the cytoplasm and non-producing clones are selected for. Furthermore, in the absence of IPTG, membrane proteins are produced at a lower rate than in the presence of IPTG. These observations indicate that in the absence of IPTG the Sec-translocon capacity is not/hardly saturated, leading to enhanced membrane protein production yields in the cytoplasmic membrane. Importantly, for more than half of the targets tested the yields obtained using un-induced BL21(DE3) cells were higher than the yields obtained in the widely used membrane protein production strains C41(DE3) and C43(DE3). Since most secretory proteins reach the periplasm via the Sec-translocon, we also monitored the production of three secretory recombinant proteins in the periplasm of BL21(DE3) cells in the presence and absence of IPTG. For all three targets tested omitting IPTG led to the highest production levels in the periplasm. Conclusions: Omission of IPTG from BL21(DE3) cells cultured in LB medium provides a very cost-and time effective alternative for the production of membrane and secretory proteins. Therefore, we recommend that this condition is incorporated in membrane- and secretory protein production screens.

Keyword
Escherichia coli, Protein production, Membrane protein, Secretory protein, BL21(DE3), T7 RNA polymerase
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-121877 (URN)10.1186/s12934-015-0328-z (DOI)000361441700007 ()26377812 (PubMedID)
Available from: 2015-10-23 Created: 2015-10-19 Last updated: 2017-12-01Bibliographically approved
4. Engineering E. coli forrhamnose PBAD promoter-based production of membrane and secretoryproteins
Open this publication in new window or tab >>Engineering E. coli forrhamnose PBAD promoter-based production of membrane and secretoryproteins
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-123416 (URN)
Available from: 2015-11-25 Created: 2015-11-25 Last updated: 2015-12-07Bibliographically approved
5. Autotransporter-Based Antigen Display in Bacterial Ghosts
Open this publication in new window or tab >>Autotransporter-Based Antigen Display in Bacterial Ghosts
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2015 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 81, no 2, 726-735 p.Article in journal (Refereed) Published
Abstract [en]

Bacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts are generated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlled production of the bacteriophage phi X174 lysis protein E. While ghosts possess all the immunostimulatory surface properties of the original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineered the Escherichia coli autotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologous proteins in Gram-negative bacteria, HbpD. Using the Mycobacterium tuberculosis vaccine target ESAT6 (early secreted antigenic target of 6 kDa), we have explored the application of HbpD to decorate E. coli and Salmonella ghosts with antigens. The use of different promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitored by determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flow cytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a protease accessibility assay, fluorescence microscopy, flow cytometry and (immuno-) electron microscopy. Here, we show that the concerted production of HbpD and lysis protein E in E. coli and Salmonella can be used to produce ghosts that efficiently display antigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsic adjuvant activity and exposure of heterologous antigens to the immune system.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-113696 (URN)10.1128/AEM.02733-14 (DOI)000347377500028 ()
Note

AuthorCount:6;

Available from: 2015-03-18 Created: 2015-02-09 Last updated: 2017-12-04Bibliographically approved

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