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High-level production of membrane proteins in E-coli BL21(DE3) by omitting the inducer IPTG
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: 7
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.

Place, publisher, year, edition, pages
2015. Vol. 14, 142
Keyword [en]
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: urn:nbn:se:su:diva-121877DOI: 10.1186/s12934-015-0328-zISI: 000361441700007PubMedID: 26377812OAI: oai:DiVA.org:su-121877DiVA: diva2:862637
Available from: 2015-10-23 Created: 2015-10-19 Last updated: 2017-12-01Bibliographically approved
In thesis
1. Optimizing membrane and secretory protein production in Gram-negative bacteria
Open this publication in new window or tab >>Optimizing membrane and secretory protein production in Gram-negative bacteria
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:nbn:se:su:diva-123418 (URN)978-91-7649-284-0 (ISBN)
Public defence
2016-01-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
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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

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