The Experts below are selected from a list of 324 Experts worldwide ranked by ideXlab platform
Roland Lloubes - One of the best experts on this subject based on the ideXlab platform.
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Interaction of the Colicin K Bactericidal Toxin with Components of Its Import Machinery in the Periplasm of Escherichia coli
Journal of bacteriology, 2010Co-Authors: Aurélie Barnéoud-arnoulet, Roland Lloubes, Marthe Gavioli, Eric CascalesAbstract:Colicins are bacterial antibiotic toxins produced by Escherichia coli cells and are active against E. coli and closely related strains. To penetrate the target cell, colicins bind to an outer membrane receptor at the cell surface and then translocate their N-terminal domain through the outer membrane and the Periplasm. Once fully translocated, the N-terminal domain triggers entry of the catalytic C-terminal domain by an unknown process. Colicin K uses the Tsx nucleoside-specific receptor for binding at the cell surface, the OmpA protein for translocation through the outer membrane, and the TolABQR proteins for the transit through the Periplasm. Here, we initiated studies to understand how the colicin K N-terminal domain (KT) interacts with the components of its transit machine in the Periplasm. We first produced KT fused to a signal sequence for Periplasm targeting. Upon production of KT in wild-type strains, cells became partly resistant to Tol-dependent colicins and sensitive to detergent, released Periplasmic proteins, and outer membrane vesicles, suggesting that KT interacts with and titrates components of its import machine. Using a combination of in vivo coimmunoprecipitations and in vitro pulldown experiments, we demonstrated that KT interacts with the TolA, TolB, and TolR proteins. For the first time, we also identified an interaction between the TolQ protein and a colicin translocation domain.
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Analysis of the Escherichia coli Tol-Pal and TonB systems by Periplasmic production of Tol, TonB, colicin, or phage capsid soluble domains.
Biochimie, 2002Co-Authors: Emmanuelle Bouveret, Laure Journet, Anne Walburger, Eric Cascales, Hélène Bénédetti, Roland LloubesAbstract:The aim of this review is to describe an in vivo assay of the interactions taking place in the Tol-Pal or TonB-ExbB-ExbD envelope complexes in the Periplasm of Escherichia coli and between them and colicins or g3p protein of filamentous bacteriophages. Domains of colicins or Periplasmic soluble domains of Tol or TonB proteins can be artificially addressed to the Periplasm of bacteria by fusing them to a signal sequence from an exported protein. These domains interact specifically in the Periplasm with the Tol or TonB complexes and disturb their function, which can be directly detected by the appearance of specific tol or tonB phenotypes. This technique can be used to detect new interactions, to characterize them biochemically and to map them or to induce tol or tonB phenotypes to study the functions of these two complexes.
Jan-willem De Gier - One of the best experts on this subject based on the ideXlab platform.
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Enhancing Recombinant Protein Yields in the E. coli Periplasm by Combining Signal Peptide and Production Rate Screening
Frontiers in microbiology, 2019Co-Authors: Alexandros Karyolaimos, Henry Ampah-korsah, Tamara Hillenaar, Anna Mestre Borras, Katarzyna Magdalena Dolata, Susanne Sievers, Katharina Riedel, Robert Daniels, Jan-willem De GierAbstract:Proteins that contain disulfide bonds mainly mature in the oxidative environment of the eukaryotic endoplasmic reticulum or the Periplasm of Gram-negative bacteria. In E. coli, disulfide bond containing recombinant proteins are often targeted to the Periplasm by an N-terminal signal peptide that is removed once it passes through the Sec-translocon in the cytoplasmic membrane. Despite their conserved targeting function, signal peptides can impact recombinant protein production yields in the Periplasm, as can the production rate. Here, we present a combined screen involving different signal peptides and varying production rates that enabled the identification of more optimal conditions for Periplasmic production of recombinant proteins with disulfide bonds. The data was generated from two targets, a single chain antibody fragment (BL1) and human growth hormone (hGH), with four different signal peptides and a titratable rhamnose promoter-based system that enables the tuning of protein production rates. Across the screen conditions, the yields for both targets significantly varied, and the optimal signal peptide and rhamnose concentration differed for each protein. Under the optimal conditions, the Periplasmic BL1 and hGH were properly folded and active. Our study underpins the importance of combinatorial screening approaches for addressing the requirements associated with the production of a recombinant protein in the Periplasm.
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Optimizing Recombinant Protein Production in the Escherichia coli Periplasm Alleviates Stress
Applied and environmental microbiology, 2018Co-Authors: Thomas Baumgarten, Roman A. Zubarev, A. Jimmy Ytterberg, Jan-willem De GierAbstract:In Escherichia coli, many recombinant proteins are produced in the Periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the Periplasm. Here, we have studied the consequences of improving the production of BL1 in the Periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is-besides the produced target-the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the Periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in E. coliIMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the Periplasm of E. coli, we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.
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Optimizing heterologous protein production in the Periplasm of E. coli by regulating gene expression levels
Microbial cell factories, 2013Co-Authors: Susan Schlegel, Edurne Rujas, Anders Jimmy Ytterberg, Roman A. Zubarev, Joen Luirink, Jan-willem De GierAbstract: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. 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. 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.
Soufian Ouchane - One of the best experts on this subject based on the ideXlab platform.
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c-Type Cytochrome Assembly Is a Key Target of Copper Toxicity within the Bacterial Periplasm.
mBio, 2015Co-Authors: Anne Durand, Asma Azzouzi, Marie-line Bourbon, Anne-soisig Steunou, Sylviane Liotenberg, Akinori Maeshima, Chantal Astier, Manuela Argentini, Shingo Saito, Soufian OuchaneAbstract:ABSTRACT In the absence of a tight control of copper entrance into cells, bacteria have evolved different systems to control copper concentration within the cytoplasm and the Periplasm. Central to these systems, the Cu + ATPase CopA plays a major role in copper tolerance and translocates copper from the cytoplasm to the Periplasm. The fate of copper in the Periplasm varies among species. Copper can be sequestered, oxidized, or released outside the cells. Here we describe the identification of CopI, a Periplasmic protein present in many proteobacteria, and show its requirement for copper tolerance in Rubrivivax gelatinosus. The Δ copI mutant is more susceptible to copper than the Cu + ATPase copA mutant. CopI is induced by copper, localized in the Periplasm and could bind copper. Interestingly, copper affects cytochrome c membrane complexes ( cbb 3 oxidase and photosystem) in both Δ copI and copA -null mutants, but the causes are different. In the copA mutant, heme and chlorophyll synthesis are affected, whereas in Δ copI mutant, the decrease is a consequence of impaired cytochrome c assembly. This impact on c -type cytochromes would contribute also to the copper toxicity in the Periplasm of the wild-type cells when they are exposed to high copper concentrations. IMPORTANCE Copper is an essential cation required as a cofactor in enzymes involved in vital processes such as respiration, photosynthesis, free radical scavenging, and pathogenesis. However, copper is highly toxic and has been implicated in disorders in all organisms, including humans, because it can catalyze the production of toxic reactive oxygen species and targets various biosynthesis pathways. Identifying copper targets, provides insights into copper toxicity and homeostatic mechanisms for copper tolerance. In this work, we describe for the first time a direct effect of excess copper on cytochrome c assembly. We show that excess copper specifically affects Periplasmic and membrane cytochromes c , thus suggesting that the copper toxicity targets c -type cytochrome biogenesis.
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c-Type Cytochrome Assembly Is a Key Target of Copper Toxicity within the Bacterial Periplasm.
mBio, 2015Co-Authors: Anne Durand, Asma Azzouzi, Marie-line Bourbon, Anne-soisig Steunou, Sylviane Liotenberg, Akinori Maeshima, Chantal Astier, Manuela Argentini, Shingo Saito, Soufian OuchaneAbstract:In the absence of a tight control of copper entrance into cells, bacteria have evolved different systems to control copper concentration within the cytoplasm and the Periplasm. Central to these systems, the Cu(+) ATPase CopA plays a major role in copper tolerance and translocates copper from the cytoplasm to the Periplasm. The fate of copper in the Periplasm varies among species. Copper can be sequestered, oxidized, or released outside the cells. Here we describe the identification of CopI, a Periplasmic protein present in many proteobacteria, and show its requirement for copper tolerance in Rubrivivax gelatinosus. The ΔcopI mutant is more susceptible to copper than the Cu(+) ATPase copA mutant. CopI is induced by copper, localized in the Periplasm and could bind copper. Interestingly, copper affects cytochrome c membrane complexes (cbb3 oxidase and photosystem) in both ΔcopI and copA-null mutants, but the causes are different. In the copA mutant, heme and chlorophyll synthesis are affected, whereas in ΔcopI mutant, the decrease is a consequence of impaired cytochrome c assembly. This impact on c-type cytochromes would contribute also to the copper toxicity in the Periplasm of the wild-type cells when they are exposed to high copper concentrations.
Eric Cascales - One of the best experts on this subject based on the ideXlab platform.
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Interaction of the Colicin K Bactericidal Toxin with Components of Its Import Machinery in the Periplasm of Escherichia coli
Journal of bacteriology, 2010Co-Authors: Aurélie Barnéoud-arnoulet, Roland Lloubes, Marthe Gavioli, Eric CascalesAbstract:Colicins are bacterial antibiotic toxins produced by Escherichia coli cells and are active against E. coli and closely related strains. To penetrate the target cell, colicins bind to an outer membrane receptor at the cell surface and then translocate their N-terminal domain through the outer membrane and the Periplasm. Once fully translocated, the N-terminal domain triggers entry of the catalytic C-terminal domain by an unknown process. Colicin K uses the Tsx nucleoside-specific receptor for binding at the cell surface, the OmpA protein for translocation through the outer membrane, and the TolABQR proteins for the transit through the Periplasm. Here, we initiated studies to understand how the colicin K N-terminal domain (KT) interacts with the components of its transit machine in the Periplasm. We first produced KT fused to a signal sequence for Periplasm targeting. Upon production of KT in wild-type strains, cells became partly resistant to Tol-dependent colicins and sensitive to detergent, released Periplasmic proteins, and outer membrane vesicles, suggesting that KT interacts with and titrates components of its import machine. Using a combination of in vivo coimmunoprecipitations and in vitro pulldown experiments, we demonstrated that KT interacts with the TolA, TolB, and TolR proteins. For the first time, we also identified an interaction between the TolQ protein and a colicin translocation domain.
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Analysis of the Escherichia coli Tol-Pal and TonB systems by Periplasmic production of Tol, TonB, colicin, or phage capsid soluble domains.
Biochimie, 2002Co-Authors: Emmanuelle Bouveret, Laure Journet, Anne Walburger, Eric Cascales, Hélène Bénédetti, Roland LloubesAbstract:The aim of this review is to describe an in vivo assay of the interactions taking place in the Tol-Pal or TonB-ExbB-ExbD envelope complexes in the Periplasm of Escherichia coli and between them and colicins or g3p protein of filamentous bacteriophages. Domains of colicins or Periplasmic soluble domains of Tol or TonB proteins can be artificially addressed to the Periplasm of bacteria by fusing them to a signal sequence from an exported protein. These domains interact specifically in the Periplasm with the Tol or TonB complexes and disturb their function, which can be directly detected by the appearance of specific tol or tonB phenotypes. This technique can be used to detect new interactions, to characterize them biochemically and to map them or to induce tol or tonB phenotypes to study the functions of these two complexes.
David P Humphreys - One of the best experts on this subject based on the ideXlab platform.
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Escherichia coli "TatExpress" strains export several g/L human growth hormone to the Periplasm by the Tat pathway
Biotechnology and bioengineering, 2019Co-Authors: Isabel Guerrero Montero, Kirsty L Richards, Chillel Jawara, Douglas F. Browning, Amber R Peswani, Mickael Labrit, Matthew Allen, Cedric Aubry, Emma Dave, David P HumphreysAbstract:Escherichia coli is a heavily used platform for the production of biotherapeutic and other high-value proteins, and a favored strategy is to export the protein of interest to the Periplasm to simplify downstream processing and facilitate disulfide bond formation. The Sec pathway is the standard means of transporting the target protein but it is unable to transport complex or rapidly folding proteins because the Sec system can only transport proteins in an unfolded state. The Tat system also operates to transport proteins to the Periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Here, we have tested the Tat system's full potential for the production of biotherapeutics for the first time using fed-batch fermentation. We expressed human growth hormone (hGH) with a Tat signal peptide in E. coli W3110 "TatExpress" strains that contain elevated levels of the Tat apparatus. This construct contained four amino acids from TorA at the hGH N-terminus as well as the initiation methionine from hGH, which is removed in vivo. We show that the protein is efficiently exported to the Periplasm during extended fed-batch fermentation, to the extent that it is by far the most abundant protein in the Periplasm. The protein was shown to be homogeneous, disulfide bonded, and active. The bioassay showed that the yields of purified Periplasmic hGH are 5.4 g/L culture whereas an enzyme-linked immunosorbent assay gave a figure of 2.39 g/L. Separate analysis of a TorA signal peptide linked to hGH construct lacking any additional amino acids likewise showed efficient export to the Periplasm, although yields were approximately two-fold lower.
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human protein disulfide isomerase functionally complements a dsba mutation and enhances the yield of pectate lyase c in escherichia coli
Journal of Biological Chemistry, 1995Co-Authors: David P Humphreys, Neil Weir, Andrew Mountain, Peter A LundAbstract:Human PDI was expressed to the Escherichia coli Periplasm, by using a plasmid encoded ompA-PDI fusion under the control of the trp promoter. Periplasmic extracts were shown to contain active PDI using the scrambled ribonuclease assay. PDI activity was also demonstrated by complementation of two phenotypes associated with a dsbA mutation. Alkaline phosphatase activity, which is reduced in dsbA cells, was restored to wild type levels by PDI. PelC, a pectate lyase from Erwinia carotovora, was shown to be DsbA dependent in E. coli. PDI was able to restore its activity to that seen in wild type cells. Increased expression of PDI was found to increase the yield of active PelC above that seen in wild type cells. PDI also enhanced the yield of PelC in DsbA- cells but only in the presence of exogenous oxidized glutathione. PDI is thus able to functionally substitute for DsbA in the folding of disulfide-bonded proteins in the bacterial Periplasm and to enhance the yield of highly expressed protein when the ability of the E. coli Periplasm to fold protein may be saturated. However, our results suggest that the activities of DsbA and PDI in vivo may be different.