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1.
Microb Cell Fact ; 17(1): 199, 2018 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-30577801

RESUMO

BACKGROUND: The secretion of recombinant disulfide-bond containing proteins into the periplasm of Gram-negative bacterial hosts, such as E. coli, has many advantages that can facilitate product isolation, quality and activity. However, the secretion machinery of E. coli has a limited capacity and can become overloaded, leading to cytoplasmic retention of product; which can negatively impact cell viability and biomass accumulation. Fine control over recombinant gene expression offers the potential to avoid this overload by matching expression levels to the host secretion capacity. RESULTS: Here we report the application of the RiboTite gene expression control system to achieve this by finely controlling cellular expression levels. The level of control afforded by this system allows cell viability to be maintained, permitting production of high-quality, active product with enhanced volumetric titres. CONCLUSIONS: The methods and systems reported expand the tools available for the production of disulfide-bond containing proteins, including antibody fragments, in bacterial hosts.


Assuntos
Expressão Gênica/genética , Transporte Proteico/genética , Proteínas Recombinantes/metabolismo
2.
Biochim Biophys Acta ; 1853(3): 756-63, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25554517

RESUMO

Numerous therapeutic proteins are expressed in Escherichia coli and targeted to the periplasm in order to facilitate purification and enable disulfide bond formation. Export is normally achieved by the Sec pathway, which transports proteins through the plasma membrane in a reduced, unfolded state. The Tat pathway is a promising alternative means of export, because it preferentially exports correctly folded proteins; however, the reducing cytoplasm of standard strains has been predicted to preclude export by Tat of proteins that contain disulfide bonds in the native state because, in the reduced state, they are sensed as misfolded and rejected. Here, we have tested a series of disulfide-bond containing biopharmaceuticals for export by the Tat pathway in CyDisCo strains that do enable disulfide bond formation in the cytoplasm. We show that interferon α2b, human growth hormone (hGH) and two antibody fragments are exported with high efficiency; surprisingly, however, they are efficiently exported even in the absence of cytoplasmic disulfide formation. The exported proteins acquire disulfide bonds in the periplasm, indicating that the normal disulfide oxidation machinery is able to act on the proteins. Tat-dependent export of hGH proceeds even when the disulfide bonds are removed by substitution of the Cys residues involved, suggesting that these substrates adopt tertiary structures that are accepted as fully-folded by the Tat machinery.


Assuntos
Dissulfetos/metabolismo , Proteínas de Escherichia coli/fisiologia , Hormônio do Crescimento Humano/metabolismo , Fragmentos de Imunoglobulinas/metabolismo , Interferon-alfa/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Periplasma/metabolismo , Sequência de Aminoácidos , Anticorpos/química , Anticorpos/metabolismo , Dissulfetos/química , Escherichia coli/metabolismo , Humanos , Interferon alfa-2 , Redes e Vias Metabólicas , Dados de Sequência Molecular , Organismos Geneticamente Modificados , Oxirredução , Transporte Proteico , Proteínas Recombinantes/metabolismo
3.
Biotechnol Prog ; 30(2): 281-90, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24376243

RESUMO

Numerous high-value therapeutic proteins are produced in Escherichia coli and exported to the periplasm, as this approach simplifies downstream processing and enables disulfide bond formation. Most recombinant proteins are exported by the Sec pathway, which transports substrates across the plasma membrane in an unfolded state. The Tat system also exports proteins to the periplasm, but transports them in a folded state. This system has attracted interest because of its tendency to transport correctly folded proteins, but this trait renders it unable to export proteins containing disulfide bonds since these are normally acquired only in the periplasm; reduced substrates tend to be recognized as incorrectly folded and rejected. In this study we have used a series of novel strains (termed CyDisCo) which oxidise disulfide bonds in the cytoplasm, and we show that these cells efficiently export a range of disulfide-containing proteins when a Tat signal peptide is attached. These test proteins include alkaline phosphatase (PhoA), a phytase containing four disulfide bonds (AppA), an antiinterleukin 1ß scFv and human growth hormone. No export of PhoA or AppA is observed in wild-type cells lacking the CyDisCo factors. The PhoA, AppA and scFv proteins were exported in an active form by Tat in the CyDisCo strain, and mass spectrometry showed that the vast majority of the scFv protein was disulfide-bonded and correctly processed. The evidence indicates that this combination of Tat + CyDisCo offers a novel means of exporting active, correctly folded disulfide bonded proteins to the periplasm.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Periplasma/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Engenharia Celular , Dissulfetos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fermentação , Proteínas de Membrana Transportadoras/genética , Periplasma/química , Dobramento de Proteína , Proteínas Recombinantes/genética
4.
FEBS J ; 280(16): 3810-21, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23745597

RESUMO

The twin-arginine translocation (Tat) system transports folded proteins across the plasma membrane in bacteria, and heterologous proteins can be exported by this pathway if a Tat-type signal peptide is present at the N-terminus. The system thus has potential for biopharmaceutical production in Escherichia coli, where export to the periplasm is often a favoured approach. Previous studies have shown that E. coli cells can export high levels of protein by the Tat pathway, and the protein product accummulates almost exclusively in the periplasm. In this study, we analysed E. coli cells that express the Bacillus subtilis TatAdCd system in place of the native TatABC system. We show that a heterologous model protein, comprising the TorA signal peptide linked to green fluorescent protein (TorA-GFP), is efficiently exported by the TatAdCd system. However, whereas the GFP is exported initially to the periplasm during batch fermentation, the mature protein is increasingly found in the extracellular culture medium. By the end of a 16-h fermentation, ~ 90% of exported GFP is present in the medium as active mature protein. The total protein profiles of the medium and periplasm are essentially identical, confirming that the outer membrane becomes leaky during the fermentation process. The cells are otherwise intact, and there is no large-scale release of cytoplasmic contents. Export levels are relatively high, with ~ 0.35 g GFP·L⁻¹ culture present in the medium. This system thus offers a means of producing recombinant protein in E. coli and harvesting directly from the medium, with potential advantages in terms of ease of purification and downstream processing.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Bactérias/genética , Técnicas de Cultura Celular por Lotes , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/ultraestrutura , Fermentação , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Proteínas de Membrana Transportadoras/genética , Oxirredutases N-Desmetilantes/biossíntese , Oxirredutases N-Desmetilantes/química , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
5.
Biotechnol Bioeng ; 109(10): 2533-42, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22539025

RESUMO

Numerous high-value recombinant proteins that are produced in bacteria are exported to the periplasm as this approach offers relatively easy downstream processing and purification. Most recombinant proteins are exported by the Sec pathway, which transports them across the plasma membrane in an unfolded state. The twin-arginine translocation (Tat) system operates in parallel with the Sec pathway but transports substrate proteins in a folded state; it therefore has potential to export proteins that are difficult to produce using the Sec pathway. In this study, we have produced a heterologous protein (green fluorescent protein; GFP) in Escherichia coli and have used batch and fed-batch fermentation systems to test the ability of the newly engineered Tat system to export this protein into the periplasm under industrial-type production conditions. GFP cannot be exported by the Sec pathway in an active form. We first tested the ability of five different Tat signal peptides to export GFP, and showed that the TorA signal peptide directed most efficient export. Under batch fermentation conditions, it was found that TorA-GFP was exported efficiently in wild type cells, but a twofold increase in periplasmic GFP was obtained when the TatABC components were co-expressed. In both cases, periplasmic GFP peaked at about the 12 h point during fermentation but decreased thereafter, suggesting that proteolysis was occurring. Typical yields were 60 mg periplasmic GFP per liter culture. The cells over-expressed the tat operon throughout the fermentation process and the Tat system was shown to be highly active over a 48 h induction period. Fed-batch fermentation generated much greater yields: using glycerol feed rates of 0.4, 0.8, and 1.2 mL h(-1), the cultures reached OD(600) values of 180 and periplasmic GFP levels of 0.4, 0.85, and 1.1 g L(-1) culture, respectively. Most or all of the periplasmic GFP was shown to be active. These export values are in line with those obtained in industrial production processes using Sec-dependent export approaches.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Engenharia Metabólica/métodos , Periplasma/metabolismo , Proteínas Recombinantes/metabolismo , Biotecnologia/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana Transportadoras/genética , Transporte Proteico , Proteínas Recombinantes/genética
6.
Biotechnol Bioeng ; 109(4): 983-91, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22125050

RESUMO

The twin arginine translocation (Tat) pathway occurs naturally in E. coli and has the distinct ability to translocate folded proteins across the inner membrane of the cell. It has the potential to export commercially useful proteins that cannot be exported by the ubiquitous Sec pathway. To better understand the bioprocess potential of the Tat pathway, this article addresses the fermentation and downstream processing performances of E. coli strains with a wild-type Tat system exporting the over-expressed substrate protein FhuD. These were compared to strains cell-engineered to over-express the Tat pathway, since the native export capacity of the Tat pathway is low. This low capacity makes the pathway susceptible to saturation by over-expressed substrate proteins, and can result in compromised cell integrity. However, there is concern in the literature that over-expression of membrane proteins, like those of the Tat pathway, can impact negatively upon membrane integrity itself. Under controlled fermentation conditions E. coli cells with a wild-type Tat pathway showed poor protein accumulation, reaching a periplasmic maximum of only 0.5 mg L⁻¹ of growth medium. Cells over-expressing the Tat pathway showed a 25% improvement in growth rate, avoided pathway saturation, and showed 40-fold higher periplasmic accumulation of FhuD. Moreover, this was achieved whilst conserving the integrity of cells for downstream processing: experimentation comparing the robustness of cells to increasing levels of shear showed no detrimental effect from pathway over-expression. Further experimentation on spheroplasts generated by the lysozyme/osmotic shock method--a scaleable way to release periplasmic protein--showed similar robustness between strains. A scale-down mimic of continuous disk-stack centrifugation predicted clarifications in excess of 90% for both intact cells and spheroplasts. Cells over-expressing the Tat pathway performed comparably to cells with the wild-type system. Overall, engineering E. coli cells to over-express the Tat pathway allowed for greater periplasmic yields of FhuD at the fermentation scale without compromising downstream processing performance.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Microbiologia Industrial/métodos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Sequência de Aminoácidos , Translocação Bacteriana , Técnicas Bacteriológicas , Transporte Biológico Ativo , Membrana Celular/metabolismo , Centrifugação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Fermentação , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/isolamento & purificação , Dados de Sequência Molecular , Periplasma/metabolismo , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/isolamento & purificação , Sinais Direcionadores de Proteínas/fisiologia , Transporte Proteico , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Esferoplastos/metabolismo , Viscosidade
7.
Biochim Biophys Acta ; 1808(3): 876-84, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21126506

RESUMO

The twin-arginine translocation (Tat) system operates in plant thylakoid membranes and the plasma membranes of most free-living bacteria. In bacteria, it is responsible for the export of a number of proteins to the periplasm, outer membrane or growth medium, selecting substrates by virtue of cleavable N-terminal signal peptides that contain a key twin-arginine motif together with other determinants. Its most notable attribute is its ability to transport large folded proteins (even oligomeric proteins) across the tightly sealed plasma membrane. In Gram-negative bacteria, TatABC subunits appear to carry out all of the essential translocation functions in the form of two distinct complexes at steady state: a TatABC substrate-binding complex and separate TatA complex. Several studies favour a model in which these complexes transiently coalesce to generate the full translocase. Most Gram-positive organisms possess an even simpler "minimalist" Tat system which lacks a TatB component and contains, instead, a bifunctional TatA component. These Tat systems may involve the operation of a TatAC complex together with a separate TatA complex, although a radically different model for TatAC-type systems has also been proposed. While bacterial Tat systems appear to require the presence of only a few proteins for the actual translocation event, there is increasing evidence for the operation of ancillary components that carry out sophisticated "proofreading" activities. These activities ensure that redox proteins are only exported after full assembly of the cofactor, thereby avoiding the futile export of apo-forms. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.


Assuntos
Arginina/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Transporte Proteico , Homologia de Sequência de Aminoácidos
8.
FEBS Lett ; 584(16): 3644-8, 2010 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-20659466

RESUMO

The twin-arginine translocation (Tat) system translocates folded proteins across biological membranes. It has been suggested that the Tat system of Escherichia coli can direct Tat substrates to degradation if they are not properly folded [Matos, C.F., Robinson, C. and Di Cola, A. (2008) The Tat system proofreads FeS protein substrates and directly initiates the disposal of rejected molecules. EMBO J. 27, 2055-2063; Matos, C.F., Di Cola, A. and Robinson, C. (2009) TatD is a central component of a Tat translocon-initiated quality control system for exported FeS proteins in Escherichia coli. EMBO Rep. 10, 474-479]. Contrary to the earlier reports, it is now concluded that reported differences between tested strains were due to variations in expression levels and inclusion body formation. Using the native Tat substrate NrfC and a malfolded variant thereof, we show that the turnover of these proteins is not affected by the absence of all known Tat components. Malfolded NrfC is degraded more quickly than the native protein, indicating that Tat-independent protease systems can recognize malfolded Tat substrates.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Substituição de Aminoácidos , Arabinose/farmacologia , Sequência de Bases , Primers do DNA/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Corpos de Inclusão/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Mutagênese Sítio-Dirigida , Dobramento de Proteína , Sinais Direcionadores de Proteínas/genética , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
9.
EMBO Rep ; 10(5): 474-9, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19343049

RESUMO

Bacterial Tat systems export folded proteins, including FeS proteins such as NrfC and NapG, which acquire their cofactors before translocation. NrfC and NapG are proofread by the Tat pathway, and misfolded examples are degraded after interaction with the translocon. Here, we identify TatD as a crucial component of this quality control system in Escherichia coli. NrfC/NapG variants lacking FeS centres are rapidly degraded in wild-type cells but stable in a DeltatatD strain. The precursor of another substrate, FhuD, is also transiently detected in wild-type cells but stable in the DeltatatD strain. Surprisingly, these substrates are stable in DeltatatD cells that overexpress TatD, and export of the non-mutated precursors is inhibited. We propose that TatD is part of a quality control system that is intimately linked to the Tat export pathway, and that the overexpression of TatD leads to an imbalance between the two systems such that both Tat-initiated turnover and export are prevented.


Assuntos
Proteínas de Escherichia coli/fisiologia , Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Arabinose/farmacologia , Cloranfenicol/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/fisiologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Oxirredutases/genética , Oxirredutases/metabolismo , Oxirredutases/fisiologia
10.
EMBO J ; 27(15): 2055-63, 2008 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-18615097

RESUMO

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane, including FeS proteins that receive their cofactors in the cytoplasm. We have studied two Escherichia coli Tat substrates, NrfC and NapG, to examine how, or whether, the system exports only correctly folded and assembled FeS proteins. With NrfC, substitutions in even one of four predicted FeS centres completely block export, indicating an effective proofreading activity. The FeS mutants are rapidly degraded but only if they interact with the Tat translocon; they are stable in a tat deletion strain and equally stable in wild-type cells if the signal peptide twin-arginine motif is removed to block targeting. Basically similar results are obtained with NapG. The Tat apparatus thus proofreads these substrates and directly initiates the turnover of rejected molecules. Turnover of mutated FeS substrates is completely dependent on the TatA/E subunits that are believed to be involved in the late stages of translocation, and we propose that partial translocation triggers substrate turnover within an integrated quality control system for FeS proteins.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas Ferro-Enxofre/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Oxirredutases/metabolismo , Sequência de Aminoácidos , Hidrolases de Éster Carboxílico/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Mutação , Dobramento de Proteína , Sinais Direcionadores de Proteínas/fisiologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Especificidade por Substrato
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