| 1. |
- Baumgarten, Thomas, et al.
(författare)
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Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived from the T7 RNA polymerase (P)-based BL21(DE3) protein production strain. In C41(DE3) and C43(DE3) mutations lowering t7rnap expression levels result in strongly reduced T7 RNAP accumulation levels. As a consequence membrane protein production stress is alleviated in the C41(DE3) and C43(DE3) strains, thereby increasing membrane protein yields. Here, we isolated Mutant56(DE3) from BL21(DE3) using a genetic screen designed to isolate BL21(DE3)-derived strains with mutations alleviating membrane protein production stress other than the ones in C41(DE3) and C43(DE3). The defining mutation of Mutant56(DE3) changes one amino acid in its T7 RNAP, which weakens the binding of the T7 RNAP to the T7 promoter governing target gene expression rather than lowering T7 RNAP levels. For most membrane proteins tested yields in Mutant56(DE3) were considerably higher than in C41(DE3) and C43(DE3). Thus, the isolation of Mutant56(DE3) shows that the evolution of BL21(DE3) can be promoted towards further enhanced membrane protein production.
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| 2. |
- Klepsch, Mirjam, 1983-, et al.
(författare)
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Immobilization of the first dimension in 2D blue native/SDS-PAGE allows the relative quantification of membrane proteomes
- 2008
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Ingår i: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 46:2, s. 48-53
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Tidskriftsartikel (refereegranskat)abstract
- In biological membranes many proteins are organized in complexes. The method of choice for the global analysis of the subunits of these complexes is two-dimensional blue native (2D BN)/SDS–PAGE. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS–PAGE. In the currently available protocols the 1st dimension BN gel lanes get distorted during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, rendering them unsuitable for comparative analysis. We have developed a 2D BN/SDS–PAGE protocol where the 1st dimension BN gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN gel lanes, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. 2D BN/SDS–PAGE with an immobilized 1st dimension was used for the comparative analysis of the cytoplasmic membrane proteomes of Escherichia coli cells overexpressing a membrane protein and to create a 2D BN/SDS–PAGE reference map of the E. coli cytoplasmic membrane proteome with 143 identified proteins from 165 different protein spots.
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| 3. |
- Schlegel, Susan, et al.
(författare)
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Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3)
- 2012
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 423:4, s. 648-659
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Schlegel, Susan, et al.
(författare)
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Revolutionizing membrane protein overexpression in bacteria
- 2010
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Ingår i: Microbial biotechnology. - : Wiley. - 1751-7915. ; 3:4, s. 403-11
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Tidskriftsartikel (refereegranskat)abstract
- The bacterium Escherichia coli is the most widely used expression host for overexpression trials of membrane proteins. Usually, different strains, culture conditions and expression regimes are screened for to identify the optimal overexpression strategy. However, yields are often not satisfactory, especially for eukaryotic membrane proteins. This has initiated a revolution of membrane protein overexpression in bacteria. Recent studies have shown that it is feasible to (i) engineer or select for E. coli strains with strongly improved membrane protein overexpression characteristics, (ii) use bacteria other than E. coli for the expression of membrane proteins, (iii) engineer or select for membrane protein variants that retain functionality but express better than the wild-type protein, and (iv) express membrane proteins using E. coli-based cell-free systems.
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| 5. |
- Wagner, Samuel, et al.
(författare)
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Tuning Escherichia coli for membrane protein overexpression
- 2008
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 105:38, s. 14371-17376
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Hjelm, Anna, et al.
(författare)
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Optimizing E. coli-Based Membrane Protein Production Using Lemo21(DE3) and GFP-Fusions
- 2013
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Ingår i: Membrane Biogenesis. - Totowa, USA : Humana Press. - 9781627034869 - 9781627034876 ; , s. 381-400
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Bokkapitel (refereegranskat)abstract
- Optimizing the conditions for the overexpression of membrane proteins in E. coli and their subsequent purification is usually a laborious and time-consuming process. Combining the Lemo21(DE3) strain, which conveniently allows to identify the optimal expression intensity of a membrane protein using only one strain, and membrane proteins C-terminally fused to Green Fluorescent Protein (GFP) greatly facilitates the production of high-quality membrane protein material for functional and structural studies.
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| 7. |
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| 8. |
- Schlegel, Jan, et al.
(författare)
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A Multiparametric and High-Throughput Platform for Host-Virus Binding Screens
- 2023
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Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6984 .- 1530-6992. ; 23:9, s. 3701-3707
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Tidskriftsartikel (refereegranskat)abstract
- Speed is key during infectious disease outbreaks. It is essential, for example, to identify critical host binding factors to pathogens as fast as possible. The complexity of host plasma membrane is often a limiting factor hindering fast and accurate determination of host binding factors as well as high-throughput screening for neutralizing antimicrobial drug targets. Here, we describe a multiparametric and high-throughput platform tackling this bottleneck and enabling fast screens for host binding factors as well as new antiviral drug targets. The sensitivity and robustness of our platform were validated by blocking SARS-CoV-2 particles with nanobodies and IgGs from human serum samples.
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| 9. |
- Schlegel, Susan, et al.
(författare)
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Bacterial-based membrane protein production
- 2014
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier BV. - 0167-4889 .- 1879-2596. ; 1843:8, s. 1739-1749
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Forskningsöversikt (refereegranskat)abstract
- Escherichia coli is by far the most widely used bacterial host for the production of membrane proteins. Usually, different strains, culture conditions and production regimes are screened for to design the optimal production process. However, these E. coli-based screening approaches often do not result in satisfactory membrane protein production yields. Recently, it has been shown that (i) E. coli strains with strongly improved membrane protein production characteristics can be engineered or selected for, (ii) many membrane proteins can be efficiently produced in E. coli-based cell-free systems, (iii) bacteria other than E. coli can be used for the efficient production of membrane proteins, and, (iv) membrane protein variants that retain functionality but are produced at higher yields than the wild-type protein can be engineered or selected for. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
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| 10. |
- Schlegel, Susan, et al.
(författare)
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De-convoluting the Genetic Adaptations of E-coli C41(DE3) in Real Time Reveals How Alleviating Protein Production Stress Improves Yields
- 2015
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 10:10, s. 1758-1766
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Tidskriftsartikel (refereegranskat)abstract
- The well-established E. coli protein production strain C41(DE3) was isolated from the T7 RNA polymerase-based BL21(DE3) strain for its ability to produce difficult recombinant proteins, and it acquired multiple mutations during its isolation. Standard allelic replacement and competition experiments were insufficient to de-convolute these mutations. By reconstructing the evolution of C41(DE3) in real time, we identified the time frames when the different mutations occurred, enabling us to link them to particular stress events. Starvation stress imposed by the isolation procedure selected for mutations enhancing nutrient uptake, and protein production stress for mutations weakening the lacUV5 promoter, which governs t7rnap expression. Moreover, recapitulating protein production stress in BL21(DE3) showed that mutations weakening the lacUV5 promoter occur through RecA-dependent recombination with the wild-type lac-promoter and are selected for upon the production of any protein. Thus, the instability of the lacUV5 promoter in BL21(DE3) alleviates protein production stress and can be harnessed to enhance production.
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| 11. |
- Schlegel, Susan, 1981-
(författare)
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From protein production to genome evolution in Escherichia coli
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 12. |
- Schlegel, Susan, et al.
(författare)
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Isolating Escherichia coli strains for recombinant protein production
- 2017
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Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Springer Science and Business Media LLC. - 1420-682X .- 1420-9071. ; 74:5, s. 891-908
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Forskningsöversikt (refereegranskat)abstract
- Escherichia coli has been widely used for the production of recombinant proteins. To improve protein production yields in E. coli, directed engineering approaches have been commonly used. However, there are only few reported examples of the isolation of E. coli protein production strains using evolutionary approaches. Here, we first give an introduction to bacterial evolution and mutagenesis to set the stage for discussing how so far selection- and screening-based approaches have been used to isolate E. coli protein production strains. Finally, we discuss how evolutionary approaches may be used in the future to isolate E. coli strains with improved protein production characteristics.
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| 13. |
- Schlegel, Susan, et al.
(författare)
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Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels
- 2013
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 12, s. 24-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 14. |
- Sych, Taras, et al.
(författare)
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High-throughput measurement of the content and properties of nano-sized bioparticles with single-particle profiler
- 2024
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Ingår i: Nature Biotechnology. - : Nature Publishing Group. - 1087-0156 .- 1546-1696. ; 42:4
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Tidskriftsartikel (refereegranskat)abstract
- Lipid nanoparticles, viruses, exosomes and liposomes are characterized by analysis of fluorescence fluctuations. We introduce a method, single-particle profiler, that provides single-particle information on the content and biophysical properties of thousands of particles in the size range 5-200 nm. We use our single-particle profiler to measure the messenger RNA encapsulation efficiency of lipid nanoparticles, the viral binding efficiencies of different nanobodies, and the biophysical heterogeneity of liposomes, lipoproteins, exosomes and viruses.
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