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From protein production to genome evolution in Escherichia coli
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of my Ph.D. studies was to improve production yields of membrane- and secretory proteins in the widely used E. coli protein production strain BL21(DE3). In this strain expression of the gene encoding the protein of interest is driven by the powerful T7 RNA polymerase (T7 RNAP) whose gene is located on the chromosome and under control of the strong, IPTG-inducible lacUV5 promoter. Unfortunately, the production of many membrane and secretory proteins is 'toxic' to BL21(DE3), resulting in poor growth and low production yields.

To understand this ‘toxicity’, the BL21(DE3) derived mutant strains C41(DE3) and C43(DE3) were characterized. Somehow, these strains can efficiently produce many ‘toxic’ membrane and secretory proteins. We showed that mutations weakening the lacUV5 promoter are responsible for this. These mutations result in a slower onset of protein production upon the addition of IPTG, which avoids saturating the Sec-translocon capacity. The Sec-translocon is a protein-conducting channel in the cytoplasmic membrane mediating the biogenesis of membrane proteins and translocation of secretory proteins. Next, we constructed a BL21(DE3)-derivative, Lemo21(DE3), in which the activity of T7 RNAP can be precisely controlled by titrating in its natural inhibitor T7 lysozyme using the rhamnose promoter system. In Lemo21(DE3), the expression level of genes encoding membrane and secretory proteins can be set such that the Sec-translocon capacity is not saturated. This is key to optimizing membrane and secretory protein production yields. Finally, reconstructing the evolution of C41(DE3) from BL21(DE3) in real time showed that during its isolation C41(DE3) had acquired mutations critical for surviving the starvation conditions used, and provided insight in how the mutations in the lacUV5 promoter had occurred.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2013. , 59 p.
Keyword [en]
Escherichia coli, BL21(DE3), protein production, membrane proteins, secretory proteins, genome evolution
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-94993ISBN: 978-91-7447-786-3 (print)OAI: oai:DiVA.org:su-94993DiVA: diva2:657543
Public defence
2013-11-22, Magnelisalen, 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 3: Manuscript.

Available from: 2013-10-30 Created: 2013-10-20 Last updated: 2013-10-29Bibliographically 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. Reconstructing the evolution of BL21 into C41 reveals rapid adaptability of a widely used E. coli strain
Open this publication in new window or tab >>Reconstructing the evolution of BL21 into C41 reveals rapid adaptability of a widely used E. coli strain
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-94992 (URN)
Available from: 2013-10-20 Created: 2013-10-20 Last updated: 2013-10-21Bibliographically approved
3. Tuning Escherichia coli for membrane protein overexpression
Open this publication in new window or tab >>Tuning Escherichia coli for membrane protein overexpression
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2008 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 38, 14371-17376 p.Article in journal (Refereed) Published
Abstract [en]

A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used “Walker strains” C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.

Keyword
engineering, systems biotechnology, proteomics
National Category
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-24888 (URN)10.1073/pnas.0804090105 (DOI)000259592400028 ()
Available from: 2008-04-30 Created: 2008-04-30 Last updated: 2013-10-21Bibliographically approved
4. Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels
Open this publication in new window or tab >>Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels
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2013 (English)In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 12, 24- p.Article in journal (Refereed) Published
Abstract [en]

Background: In Escherichia coli many heterologous proteins are produced in the periplasm. To direct these proteins to the periplasm, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec-translocon. For poorly understood reasons, the production of heterologous secretory proteins is often toxic to the cell thereby limiting yields. To gain insight into the mechanism(s) that underlie this toxicity we produced two secretory heterologous proteins, super folder green fluorescent protein and a single-chain variable antibody fragment, in the Lemo21(DE3) strain. In this strain, the expression intensity of the gene encoding the target protein can be precisely controlled. Results: Both SFGFP and the single-chain variable antibody fragment were equipped with a DsbA-derived signal sequence. Producing these proteins following different gene expression levels in Lemo21(DE3) allowed us to identify the optimal expression level for each target gene. Too high gene expression levels resulted in saturation of the Sec-translocon capacity as shown by hampered translocation of endogenous secretory proteins and a protein misfolding/aggregation problem in the cytoplasm. At the optimal gene expression levels, the negative effects of the production of the heterologous secretory proteins were minimized and yields in the periplasm were optimized. Conclusions: Saturating the Sec-translocon capacity can be a major bottleneck hampering heterologous protein production in the periplasm. This bottleneck can be alleviated by harmonizing expression levels of the genes encoding the heterologous secretory proteins with the Sec-translocon capacity. Mechanistic insight into the production of proteins in the periplasm is key to optimizing yields in this compartment.

Keyword
Recombinant protein, Protein production, Escherichia coli, Lemo21(DE3), Protein translocation, Periplasm, Sec-translocon
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-89590 (URN)10.1186/1475-2859-12-24 (DOI)000316339500001 ()
Note

AuthorCount:6;

Available from: 2013-04-30 Created: 2013-04-30 Last updated: 2017-12-06Bibliographically approved

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