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Enhancing membrane and secretory protein production yields in Escherichia coli
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins fulfill essential functions in living cells. To produce sufficient amounts of a protein is essential to study the structure and function of a protein, or to use it for medical purposes. Escherichia coli is a Gram-negative bacterium that is widely used for recombinant protein production. The aim of my PhD studies was to enhance membrane and secretory protein production yields using E. coli. The T7-based protein production system BL21(DE3)/pET was mainly used in my studies. BL21(DE3) contains a strong IPTG-inducible lacUV5 promoter governing the expression of the t7rnap gene encoding the T7RNAP on its chromosome. The target gene is under control of the T7 promoter on the pET plasmid. T7RNAP specifically recognizes the T7 promoter and transcribes the target gene more efficiently than the bacterial RNAP. Unfortunately, the biogenesis machinery for membrane and secretory proteins is usually saturated by the high protein production intensity when the BL21(DE3)/pET system is induced with IPTG, thereby negatively affecting protein production yields. In the first study, we found that when using the BL21(DE3)/pET system omitting the inducer IPTG improved membrane and secretory protein production yields. In previous studies, Lemo21(DE3) was developed to facilitate the production of membrane and secretory proteins. Lemo21(DE3) contains the pLemo plasmid in which the gene encoding the inhibitor of T7RNAP, T7 lysozyme, is under the control of the rhaBAD promoter. The activity of T7RNAP is regulated by synthesizing different levels of T7 lysozyme by adding different amounts of rhamnose. Thus, the production intensity can be modulated such that the biogenesis machinery of membrane and secretory proteins is not saturated upon IPTG induction. In the second study, we combined the key elements from both the pLemo and pET vectors to create the pReX (Regulated eXpression) plasmid to facilitate the use of helper plasmids encoding e.g., chaperones when it is necessary. In the third study, we used the rhaBAD promoter to direct the production of membrane and secretory proteins in a rhamnose metabolism and active uptake deficient strain. The protein production rate can be truly tuned in this setup. Therefore, the production of membrane and secretory proteins can be enhanced by using the right amount of rhamnose in the culture medium. BL21(DE3) contains the λDE3 prophage that carries the t7rnap gene under the control of the lacUV5 promoter. The λDE3 prophage is thought to be stably inserted into the chromosome, but the lytic cycle of the prophage can still be induced by the SOS response inducing antibiotic mitomycin C in the mitomycin C-based bacteriophage test. In the fourth study, we engineered BL21T7 by deleting in BL21(DE3) lysis related genes from the prophage. BL21T7 has similar recombinant protein production characteristics as its ancestor BL21(DE3).

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2020. , p. 71
Keywords [en]
recombinant protein production, Escherichia coli, membrane protein, secretory protein, strain engineering
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-176362ISBN: 978-91-7797-923-4 (print)ISBN: 978-91-7797-924-1 (electronic)OAI: oai:DiVA.org:su-176362DiVA, id: diva2:1375693
Public defence
2020-02-06, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 09:30 (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: 2020-01-14 Created: 2019-12-05 Last updated: 2019-12-18Bibliographically approved
List of papers
1. 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, article id 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.

Keywords
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: 2019-12-05Bibliographically approved
2. The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coli
Open this publication in new window or tab >>The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coli
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2017 (English)In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 16, article id 226Article in journal (Refereed) Published
Abstract [en]

Background: To optimize the production of membrane and secretory proteins in Escherichia coli, it is critical to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein production strain. In Lemo21(DE3), the T7 RNA polymerase activity can be modulated by the controlled co-production of its natural inhibitor T7 lysozyme. This setup enables to precisely tune target gene expression rates in Lemo21(DE3). The t7lys gene is expressed from the pLemo plasmid using the titratable rhamnose promoter. A disadvantage of the Lemo21(DE3) setup is that the system is based on two plasmids, a T7 expression vector and pLemo. The aim of this study was to simplify the Lemo21(DE3) setup by incorporating the key elements of pLemo in a standard T7-based expression vector.

Results: By incorporating the gene encoding the T7 lysozyme under control of the rhamnose promoter in a standard T7-based expression vector, pReX was created (ReX stands for Regulated gene eXpression). For two model membrane proteins and a model secretory protein we show that the optimized production yields obtained with the pReX expression vector in BL21(DE3) are similar to the ones obtained with Lemo21(DE3) using a standard T7 expression vector. For another secretory protein, a c-type cytochrome, we show that pReX, in contrast to Lemo21(DE3), enables the use of a helper plasmid that is required for the maturation and hence the production of this heme c protein.

Conclusions: Here, we created pReX, a T7-based expression vector that contains the gene encoding the T7 lysozyme under control of the rhamnose promoter. pReX enables regulated T7-based target gene expression using only one plasmid. We show that with pReX the production of membrane and secretory proteins can be readily optimized. Importantly, pReX facilitates the use of helper plasmids. Furthermore, the use of pReX is not restricted to BL21(DE3), but it can in principle be used in any T7 RNAP-based strain. Thus, pReX is a versatile alternative to Lemo21(DE3).

Keywords
Escherichia coli, Protein production, Membrane protein, Secretory protein, T7 RNA polymerase, Lemo21(DE3)
National Category
Environmental Biotechnology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-150956 (URN)10.1186/s12934-017-0840-4 (DOI)000418086100001 ()29246156 (PubMedID)
Available from: 2018-01-12 Created: 2018-01-12 Last updated: 2019-12-06Bibliographically approved
3. Tailoring Escherichia coli for the L-Rhamnose P-BAD Promoter-Based Production of Membrane and Secretory Proteins
Open this publication in new window or tab >>Tailoring Escherichia coli for the L-Rhamnose P-BAD Promoter-Based Production of Membrane and Secretory Proteins
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2017 (English)In: ACS Photonics, E-ISSN 2330-4022, Vol. 6, no 6, p. 985-994Article in journal (Refereed) Published
Abstract [en]

Membrane and secretory protein production in Escherichia coli requires precisely controlled production rates to avoid the deleterious saturation of their biogenesis pathways. On the basis of this requirement, the E. coli L-rhamnose PBAD promoter (PrhaBAD) is often used for membrane and secretory protein production since PrhaBAD is thought to regulate protein production rates in an L-rhamnose concentration-dependent manner. By monitoring protein production in real-time in E. coli wild-type and an L-rhamnose catabolism deficient mutant, we demonstrate that the L-rhamnose concentration-dependent tunability of PrhaBAD-mediated protein production is actually due to L-rhamnose consumption rather than regulating production rates. Using this information, a RhaT-mediated L-rhamnose transport and L-rhamnose catabolism deficient double mutant was constructed. We show that this mutant enables the regulation of PrhaBAD-based protein production rates in an L-rhamnose concentration-dependent manner and that this is critical to optimize membrane and secretory protein production yields. The high precision of protein production rates provided by the PrhaBAD promoter in an L-rhamnose transport and catabolism deficient background could also benefit other applications in synthetic biology.

Keywords
E. coli, protein production, L-rhamnose promoter, L-rhamnose metabolism, membrane protein, secretory protein
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-145244 (URN)10.1021/acssynbio.6b00321 (DOI)000403864900008 ()28226208 (PubMedID)
Available from: 2017-07-27 Created: 2017-07-27 Last updated: 2019-12-06Bibliographically approved
4. Construction and characterization of the bacteriophage testable BL21(DE3)-derivative BL21T7
Open this publication in new window or tab >>Construction and characterization of the bacteriophage testable BL21(DE3)-derivative BL21T7
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-176359 (URN)
Available from: 2019-12-03 Created: 2019-12-03 Last updated: 2019-12-05Bibliographically approved

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