RESUMO
Influenza A viruses and the bacterium Streptococcus pneumoniae (pneumococci) both express neuraminidases that catalyze release of sialic acid residues from oligosaccharides and glycoproteins. Although these respiratory pathogen neuraminidases function in a similar environment, it remains unclear if these enzymes use similar mechanisms for sialic acid cleavage. Here, we compared the enzymatic properties of neuraminidases from two influenza A subtypes (N1 and N2) and the pneumococcal strain TIGR4 (NanA, NanB, and NanC). Insect cell-produced N1 and N2 tetramers exhibited calcium-dependent activities and stabilities that varied with pH. In contrast, E. coli-produced NanA, NanB, and NanC were isolated as calcium insensitive monomers with stabilities that were more resistant to pH changes. Using a synthetic substrate (MUNANA), all neuraminidases showed similar pH optimums (pH 6-7) that were primarily defined by changes in catalytic rate rather than substrate binding affinity. Upon using a multivalent substrate (fetuin sialoglycans), much higher specific activities were observed for pneumococcal neuraminidases that contain an additional lectin domain. In virions, N1 and especially N2 also showed enhanced specific activity toward fetuin that was lost upon the addition of detergent, indicating the sialic acid-binding capacity of neighboring hemagglutinin molecules likely contributes to catalysis of natural multivalent substrates. These results demonstrate that influenza and pneumococcal neuraminidases have evolved similar yet distinct strategies to optimize their catalytic activity.
Assuntos
Vírus da Influenza A , Ácido N-Acetilneuramínico , Neuraminidase , Cálcio/metabolismo , Catálise , Escherichia coli/enzimologia , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/metabolismo , Streptococcus pneumoniae/enzimologia , Vírus da Influenza A/enzimologia , Animais , Linhagem CelularRESUMO
Main reasons to produce recombinant proteins in the periplasm of E. coli rather than in its cytoplasm are to -i- enable disulfide bond formation, -ii- facilitate protein isolation, -iii- control the nature of the N-terminus of the mature protein, and -iv- minimize exposure to cytoplasmic proteases. However, hampered protein targeting, translocation and folding as well as protein instability can all negatively affect periplasmic protein production yields. Strategies to enhance periplasmic protein production yields have focused on harmonizing secretory recombinant protein production rates with the capacity of the secretory apparatus by transcriptional and translational tuning, signal peptide selection and engineering, increasing the targeting, translocation and periplasmic folding capacity of the production host, preventing proteolysis, and, finally, the natural and engineered adaptation of the production host to periplasmic protein production. Here, we discuss these strategies using notable examples as a thread.
RESUMO
Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond-containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild-type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding-induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide-stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra-cytosolic compartment, like the periplasm.
Assuntos
Fosfatase Alcalina/biossíntese , Proteínas de Escherichia coli/biossíntese , Escherichia coli/enzimologia , Periplasma/enzimologia , Biossíntese de Proteínas , Dobramento de Proteína , Fosfatase Alcalina/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Periplasma/genéticaRESUMO
N-linked glycans commonly contribute to secretory protein folding, sorting, and signaling. For enveloped viruses, such as the influenza A virus (IAV), large N-linked glycans can also be added to prevent access to epitopes on the surface antigens hemagglutinin (HA or H) and neuraminidase (NA or N). Sequence analysis showed that in the NA head domain of H1N1 IAVs, three N-linked glycosylation sites are conserved and that a fourth site is conserved in H3N2 IAVs. Variable sites are almost exclusive to H1N1 IAVs of human origin, where the number of head glycosylation sites first increased over time and then decreased with and after the introduction of the 2009 pandemic H1N1 IAV of Eurasian swine origin. In contrast, variable sites exist in H3N2 IAVs of human and swine origin, where the number of head glycosylation sites has mainly increased over time. Analysis of IAVs carrying N1 and N2 mutants demonstrated that the N-linked glycosylation sites on the NA head domain are required for efficient virion incorporation and replication in cells and eggs. It also revealed that N1 stability is more affected by the head domain glycans, suggesting N2 is more amenable to glycan additions. Together, these results indicate that in addition to antigenicity, N-linked glycosylation sites can alter NA enzymatic stability and the NA amount in virions.IMPORTANCE N-linked glycans are transferred to secretory proteins upon entry into the endoplasmic reticulum lumen. In addition to promoting secretory protein maturation, enveloped viruses also utilize these large oligosaccharide structures to prevent access to surface antigen epitopes. Sequence analyses of the influenza A virus (IAV) surface antigen neuraminidase (NA or N) showed that the conservation of N-linked glycosylation sites on the NA enzymatic head domain differs by IAV subtype (H1N1 versus H3N2) and species of origin, with human-derived IAVs possessing the most variability. Experimental analyses verified that the N-linked glycosylation sites on the NA head domain contribute to virion incorporation and replication. It also revealed that the head domain glycans affect N1 stability more than N2, suggesting N2 is more accommodating to glycan additions. These results demonstrate that in addition to antigenicity, changes in N-linked glycosylation sites can alter other properties of viral surface antigens and virions.
Assuntos
Vírus da Influenza A/metabolismo , Neuraminidase/química , Neuraminidase/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Replicação Viral/fisiologia , Animais , Antígenos Virais/metabolismo , Linhagem Celular , Cães , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H3N2/metabolismo , Vírus da Influenza A/genética , Células Madin Darby de Rim Canino , Modelos Moleculares , Mutação , Neuraminidase/genética , Dobramento de Proteína , Suínos , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/metabolismoRESUMO
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.
RESUMO
Escherichia coli has been widely used as a platform microorganism for both membrane protein production and cell factory engineering. The current methods to produce membrane proteins in this organism require the induction of target gene expression and often result in unstable, low yields. Here, we present a method combining a constitutive promoter with a library of bicistronic design (BCD) elements, which enables inducer-free, tuned translation initiation for optimal protein production. Our system mediates stable, constitutive production of bacterial membrane proteins at yields that outperform those obtained with E. coli Lemo21(DE3), the current gold standard for bacterial membrane protein production. We envisage that the continuous, fine-tunable, and high-level production of membrane proteins by our method will greatly facilitate their study and their utilization in engineering cell factories.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteínas de Membrana/genética , Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/genética , Vetores Genéticos/genética , Regiões Promotoras Genéticas/genéticaRESUMO
Whereas, bacterial inclusion bodies (IBs) for long were regarded as undesirable aggregates emerging during recombinant protein production, they currently receive attention as promising nanoparticulate biomaterials with diverse applications in biotechnology and biomedicine. We previously identified ssTorA, a signal sequence that normally directs protein export via the Tat pathway in E. coli, as a tag that induces the accumulation of fused proteins into IBs under overexpression conditions. Here, we used targeted mutagenesis to identify features and motifs being either critical or dispensable for IB formation. We found that IB formation is neither related to the function of ssTorA as a Tat-signal sequence nor is it a general feature of this family of signal sequences. IB formation was inhibited by co-overexpression of ssTorA binding chaperones TorD and DnaK and by amino acid substitutions that affect the propensity of ssTorA to form an α-helix. Systematic deletion experiments identified a minimal region of ssTorA required for IB formation in the center of the signal sequence. Unbiased genetic screening of a library of randomly mutagenized ssTorA sequences for reduced aggregation properties allowed us to pinpoint residues that are critical to sustain insoluble expression. Together, the data point to possible mechanisms for the aggregation of ssTorA fusions. Additionally, they led to the design of a tag with superior IB-formation properties compared to the original ssTorA sequence.
RESUMO
Recently, we engineered a tunable rhamnose promoter-based setup for the production of recombinant proteins in E. coli. This setup enabled us to show that being able to precisely set the production rate of a secretory recombinant protein is critical to enhance protein production yields in the periplasm. It is assumed that precisely setting the production rate of a secretory recombinant protein is required to harmonize its production rate with the protein translocation capacity of the cell. Here, using proteome analysis we show that enhancing periplasmic production of human Growth Hormone (hGH) using the tunable rhamnose promoter-based setup is accompanied by increased accumulation levels of at least three key players in protein translocation; the peripheral motor of the Sec-translocon (SecA), leader peptidase (LepB), and the cytoplasmic membrane protein integrase/chaperone (YidC). Thus, enhancing periplasmic hGH production leads to increased Sec-translocon capacity, increased capacity to cleave signal peptides from secretory proteins and an increased capacity of an alternative membrane protein biogenesis pathway, which frees up Sec-translocon capacity for protein secretion. When cells with enhanced periplasmic hGH production yields were harvested and subsequently cultured in the absence of inducer, SecA, LepB, and YidC levels went down again. This indicates that when using the tunable rhamnose-promoter system to enhance the production of a protein in the periplasm, E. coli can adapt its protein translocation machinery for enhanced recombinant protein production in the periplasm.
RESUMO
Many bacteria export effector proteins fulfilling their function in membranes of a eukaryotic host. These effector membrane proteins appear to contain signals for two incompatible bacterial secretion pathways in the same protein: a specific export signal, as well as transmembrane segments that one would expect to mediate targeting to the bacterial inner membrane. Here, we show that the transmembrane segments of effector proteins of type III and type IV secretion systems indeed integrate in the membrane as required in the eukaryotic host, but that their hydrophobicity in most instances is just below the threshold required for mediating targeting to the bacterial inner membrane. Furthermore, we show that binding of type III secretion chaperones to both the effector's chaperone-binding domain and adjacent hydrophobic transmembrane segments also prevents erroneous targeting. These results highlight the evolution of a fine discrimination between targeting pathways that is critical for the virulence of many bacterial pathogens.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Proteínas de Membrana/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Ligação Proteica , VirulênciaRESUMO
The bacterium Escherichia coli has been widely used for the production of both pro- and eukaryotic membrane proteins. Usually, a set of standard strains as well as different culture setups and induction regimes are screened for to enhance production yields. However, on a limited scale, E. coli strains have been isolated for recombinant helical bundle membrane protein production using both selection- and engineering-based approaches. Here, we discuss how such approaches have been used so far to shape E. coli for the production of these recombinant membrane proteins and may be used in the future to further enhance production yields.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Recombinantes/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Engenharia Genética , Proteínas de Membrana/genética , Proteínas Recombinantes/genéticaRESUMO
Vitamin B12 (cobalamin) is the most complex B-type vitamin and is synthetized exclusively in a limited number of prokaryotes. Its biologically active variants contain rare organometallic bonds, which are used by enzymes in a variety of central metabolic pathways such as L-methionine synthesis and ribonucleotide reduction. Although its biosynthesis and role as co-factor are well understood, knowledge about uptake of cobalamin by prokaryotic auxotrophs is scarce. Here, we characterize a cobalamin-specific ECF-type ABC transporter from Lactobacillus delbrueckii, ECF-CbrT, and demonstrate that it mediates the specific, ATP-dependent uptake of cobalamin. We solved the crystal structure of ECF-CbrT in an apo conformation to 3.4 Å resolution. Comparison with the ECF transporter for folate (ECF-FolT2) from the same organism, reveals how the identical ECF module adjusts to interact with the different substrate binding proteins FolT2 and CbrT. ECF-CbrT is unrelated to the well-characterized B12 transporter BtuCDF, but their biochemical features indicate functional convergence.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Apoproteínas/química , Proteínas de Bactérias/química , Ácido Fólico/química , Lactobacillus delbrueckii/química , Vitamina B 12/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Apoproteínas/genética , Apoproteínas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Transporte Biológico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Fólico/metabolismo , Expressão Gênica , Teste de Complementação Genética , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Lactobacillus delbrueckii/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Vitamina B 12/metabolismoRESUMO
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.
Assuntos
Escherichia coli/genética , Periplasma/metabolismo , Proteínas Recombinantes/biossíntese , Anticorpos de Cadeia Única/biossíntese , Estresse Fisiológico , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Sinais Direcionadores de Proteínas/genética , Transporte Proteico , Proteoma , Proteômica , Proteínas Recombinantes/genética , Anticorpos de Cadeia Única/genéticaRESUMO
Tail-anchored membrane proteins (TAMPs) are relatively simple membrane proteins characterized by a single transmembrane domain (TMD) at their C-terminus. Consequently, the hydrophobic TMD, which acts as a subcellular targeting signal, emerges from the ribosome only after termination of translation precluding canonical co-translational targeting and membrane insertion. In contrast to the well-studied eukaryotic TAMPs, surprisingly little is known about the cellular components that facilitate the biogenesis of bacterial TAMPs. In this study, we identify DjlC and Flk as bona fide Escherichia coli TAMPs and show that their TMDs are necessary and sufficient for authentic membrane targeting of the fluorescent reporter mNeonGreen. Using strains conditional for the expression of known E. coli membrane targeting and insertion factors, we demonstrate that the signal recognition particle (SRP), its receptor FtsY, the chaperone DnaK and insertase YidC are each required for efficient membrane localization of both TAMPs. A close association between the TMD of DjlC and Flk with both the Ffh subunit of SRP and YidC was confirmed by site-directed in vivo photo-crosslinking. In addition, our data suggest that the hydrophobicity of the TMD correlates with the dependency on SRP for efficient targeting.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Proteínas de Bactérias/análise , Escherichia coli/citologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/análise , Proteínas de Choque Térmico HSP70/análise , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana Transportadoras/análise , Receptores Citoplasmáticos e Nucleares/análise , Partícula de Reconhecimento de Sinal/análiseRESUMO
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).
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos/genética , Proteínas de Membrana/genética , Proteínas Virais/metabolismo , Clonagem Molecular , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/metabolismo , Vetores Genéticos/metabolismo , Proteínas de Membrana/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Transporte Proteico , Proteínas Virais/genéticaRESUMO
Optimizing the conditions for the production of membrane proteins in E. coli is usually a laborious and time-consuming process. Combining the Lemo21(DE3) strain or the pReX T7-based expression vector with membrane proteins C-terminally fused to Green Fluorescent Protein (GFP) greatly facilitates the optimization of membrane protein production yields. Both Lemo21(DE3) and pReX allow precise regulation of expression intensities of genes encoding membrane proteins, which is critical to identify the optimal production condition for a membrane protein. The use of GFP-fusions allows direct monitoring and visualization of membrane proteins at any stage during the production optimization process.
Assuntos
Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Membrana/genética , Animais , Técnicas de Cultura de Células , Clonagem Molecular/métodos , Expressão Gênica , Vetores Genéticos/genética , Humanos , Proteínas Recombinantes de Fusão/genética , Transformação GenéticaRESUMO
BACKGROUND: Heterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host. Production of proteins in an insoluble form in inclusion bodies (IBs) can alleviate these problems. Unfortunately, the propensity of heterologous proteins to form IBs is variable and difficult to predict. Hence, fusing the target protein to an aggregation prone polypeptide or IB-tag is a useful strategy to produce difficult-to-express proteins in an insoluble form. RESULTS: When screening for signal sequences that mediate optimal targeting of heterologous proteins to the periplasmic space of E. coli, we observed that fusion to the 39 amino acid signal sequence of E. coli TorA (ssTorA) did not promote targeting but rather directed high-level expression of the human proteins hEGF, Pla2 and IL-3 in IBs. Further analysis revealed that ssTorA even mediated IB formation of the highly soluble endogenous E. coli proteins TrxA and MBP. The ssTorA also induced aggregation when fused to the C-terminus of target proteins and appeared functional as IB-tag in E. coli K-12 as well as B strains. An additive effect on IB-formation was observed upon fusion of multiple ssTorA sequences in tandem, provoking almost complete aggregation of TrxA and MBP. The ssTorA-moiety was successfully used to produce the intrinsically unstable hEGF and the toxic fusion partner SymE, demonstrating its applicability as an IB-tag for difficult-to-express and toxic proteins. CONCLUSIONS: We present proof-of-concept for the use of ssTorA as a small, versatile tag for robust E. coli-based expression of heterologous proteins in IBs.
Assuntos
Escherichia coli/genética , Corpos de Inclusão/metabolismo , Interleucina-3/biossíntese , Sinais Direcionadores de Proteínas/genética , Proteínas de Transporte , Fator de Crescimento Epidérmico/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Vetores Genéticos , Humanos , Corpos de Inclusão/química , Interleucina-3/genética , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Solubilidade , Tiorredoxinas/química , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
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.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Membrana/metabolismo , Mutação , Proteínas Virais/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Regiões Promotoras Genéticas , Proteínas Virais/metabolismoRESUMO
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.
Assuntos
Escherichia coli/genética , Proteínas de Membrana/genética , Engenharia Metabólica/métodos , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/genética , Ramnose/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Escherichia coli/isolamento & purificação , Proteínas Recombinantes/biossíntese , Evolução Biológica , DNA Bacteriano/metabolismo , Mutagênese , Mutação/genéticaRESUMO
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-ß-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.