RESUMO
The success of using the insect cell-baculovirus expression technology (BEST) relies on the efficient construction of recombinant baculovirus with genetic stability and high productivity, ideally within a short time period. Generation of recombinant baculoviruses requires the transfection of insect cells, harvesting of recombinant baculovirus pools, isolation of plaques, and the expansion of baculovirus stocks for their use for recombinant protein production. Moreover, many options exist for selecting the genetic elements to be present in the recombinant baculovirus. This chapter describes the most commonly used homologous recombination systems for the production of recombinant baculoviruses, as well as strategies to maximize generation efficiency and recombinant protein or baculovirus production. The key steps for generating baculovirus stocks and troubleshooting strategies are described.
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Baculoviridae , Proteínas Recombinantes , Baculoviridae/genética , Animais , Proteínas Recombinantes/genética , Vetores Genéticos/genética , Transfecção/métodos , Recombinação Homóloga , Células Sf9 , Linhagem Celular , Spodoptera/virologia , Insetos/genética , Insetos/virologiaRESUMO
Virus-like particles (VLP) of the cowpea chlorotic mottle virus (CCMV), a plant virus, have been shown to be safe and noncytotoxic vehicles for delivering various cargos, including nucleic acids and peptides, and as scaffolds for presenting epitopes. Thus, CCMV-VLP have acquired increasing attention to be used in fields such as gene therapy, drug delivery, and vaccine development. Regardless of their production method, most reports purify CCMV-VLP through a series of ultracentrifugation steps using sucrose density gradient ultracentrifugation, which is a complex and time-consuming process. Here, the use of anion exchange chromatography is described as a one-step protocol for purification of CCMV-VLP produced by the insect cell-baculovirus expression vector system (IC-BEVS).
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Bromovirus , Bromovirus/genética , Animais , Baculoviridae/genética , Vetores Genéticos/genética , Cromatografia por Troca Iônica/métodos , Vírion/isolamento & purificação , Vírion/genética , Vírion/metabolismoRESUMO
BACKGROUND: Developing effective vaccines against SARS-CoV-2 that consider manufacturing limitations, equitable access, and acceptance is necessary for developing platforms to produce antigens that can be efficiently presented for generating neutralizing antibodies and as a model for new vaccines. RESULTS: This work presents the development of an applicable technology through the oral administration of the SARS-CoV-2 RBD antigen fused with a peptide to improve its antigenic presentation. We focused on the development and production of the recombinant receptor binding domain (RBD) produced in E. coli modified with the addition of amino acids extension designed to improve antigen presentation. The production was carried out in shake flask and bioreactor cultures, obtaining around 200 mg/L of the antigen. The peptide-fused RBD and peptide-free RBD proteins were characterized and compared using SDS-PAGE gel, high-performance chromatography, and circular dichroism. The peptide-fused RBD was formulated in an oil-in-water emulsion for oral mice immunization. The peptide-fused RBD, compared to RBD, induced robust IgG production in mice, capable of recognizing the recombinant RBD in Enzyme-linked immunosorbent assays. In addition, the peptide-fused RBD generated neutralizing antibodies in the sera of the dosed mice. The formulation showed no reactive episodes and no changes in temperature or vomiting. CONCLUSIONS: Our study demonstrated the effectiveness of the designed peptide added to the RBD to improve antigen immunostimulation by oral administration.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Humanos , Camundongos , Adjuvantes Imunológicos , Vacinas contra COVID-19 , Escherichia coli , Administração Oral , Antígenos Virais , Anticorpos Neutralizantes , Peptídeos , Anticorpos AntiviraisRESUMO
A significant problem during recombinant protein production is proteolysis. One of the most common preventive strategies is the addition of protease inhibitors, which has drawbacks, such as their short half-life and high cost, and their limited prevention of extracellular proteolysis. Actinomycetes produce the most commonly used inhibitors, which are non-ribosomal small aldehydic peptides. Previously, an unprecedented biosynthetic route involving a condensation-minus non-ribosomal peptide synthetase (NRPSs) and a tRNA utilizing enzyme (tRUE) was shown to direct the synthesis of one of these inhibitor peptides, livipeptin. Here, we show that expression of the livipeptin biosynthetic pathway encoded by the lvp genes in CHO cells resulted in the production of this metabolite with cysteine protease inhibitory activity, implying that mammalian tRNAs were recruited by the lvp system. CHO cells transiently expressing the biosynthetic pathway produced livipeptin without affecting cell growth or viability. Expression of the lvp system in CHO cells producing two model proteins, secreted alkaline phosphatase (hSeAP) and a monoclonal antibody, resulted in higher specific productivity with reduced proteolysis. We show for the first time that the expression of a bacterial biosynthetic pathway is functional in CHO cells, resulting in the efficient, low-cost synthesis of a protease inhibitor without adverse effects on CHO cells. This expands the field of metabolic engineering of mammalian cells by expressing the overwhelming diversity of actinomycetes biosynthetic pathways and opens a new option for proteolysis inhibition in bioprocess engineering.
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Vias Biossintéticas , Peptídeos , Cricetinae , Animais , Cricetulus , Proteólise , Células CHO , Proteínas RecombinantesRESUMO
Vaccine development against dengue virus is challenging because of the antibody-dependent enhancement of infection (ADE), which causes severe disease. Consecutive infections by Zika (ZIKV) and/or dengue viruses (DENV), or vaccination can predispose to ADE. Current vaccines and vaccine candidates contain the complete envelope viral protein, with epitopes that can raise antibodies causing ADE. We used the envelope dimer epitope (EDE), which induces neutralizing antibodies that do not elicit ADE, to design a vaccine against both flaviviruses. However, EDE is a discontinuous quaternary epitope that cannot be isolated from the E protein without other epitopes. Utilizing phage display, we selected three peptides that mimic the EDE. Free mimotopes were disordered and did not elicit an immune response. After their display on adeno-associated virus (AAV) capsids (VLP), they recovered their structure and were recognized by an EDE-specific antibody. Characterization by cryo-EM and enzyme-linked immunosorbent assay confirmed the correct display of a mimotope on the surface of the AAV VLP and its recognition by the specific antibody. Immunization with the AAV VLP displaying one of the mimotopes induced antibodies that recognized ZIKV and DENV. This work provides the basis for developing a Zika and dengue virus vaccine candidate that will not induce ADE.
Assuntos
Vírus da Dengue , Dengue , Vacinas , Infecção por Zika virus , Zika virus , Humanos , Infecção por Zika virus/prevenção & controle , Vírus da Dengue/química , Dengue/prevenção & controle , Anticorpos Antivirais , Proteínas do Envelope Viral/química , Anticorpos Neutralizantes , Epitopos , Reações CruzadasRESUMO
A major challenge for developing countries during the COVID-19 pandemic is affordable and adequate monitoring of disease progression and population exposure as the primary source relevant epidemiological indicators. Serology testing enables assessing population exposure and to guide vaccination strategies but requires rigorous accuracy validation before population-wide implementation. We adapted a two-step ELISA protocol as a single-step protocol for detection of IgG against the Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein and compared its diagnostic accuracy with a commercial immunoassay anti-nucleoprotein IgG. Both methods yielded adequate and comparable diagnostic accuracy after 3 weeks post-symptom onset and were implemented in a nation-wide population based serological survey during August-November 2020. Anti-RBD National seroprevalence was 23.6%, 1.3% lower, but not significantly, than for anti-N. Double positive seroprevalence was 19.7%. Anti-N single-positive seroprevalence was 3.72% and anti-RBD single-positive seroprevalence was 1.98%. Discrepancies in the positivity to either single marker may be due to different kinetics of each antibody marker as well as the heterogeneity of the sampling time in regards to local epidemic waves. Baseline single positivity prevalence will be useful to assess the serological impact of vaccination and natural infection in further serosurveillance efforts.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Anticorpos Antivirais , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/prevenção & controle , Imunoglobulina G , México/epidemiologia , Pandemias , Estudos Soroepidemiológicos , VacinaçãoRESUMO
Sericin, a silk-derived non-immunogenic protein, has been used to improve cell culture performance by increasing viability, cell concentration, and promoting adherence of several cell lines. Here, we hypothesized that the properties of sericin can enhance the amplification of flaviviruses in cell cultures. The propagation of flavivirus is inefficient and limits scientific research. Zika virus (ZIKV) is an important human pathogen that has been widely studied because of its high impact on public health. There is a need to amplify Zika virus both for research and vaccine development. In this work, we show that sericin improves ZIKV amplification in insect (C6/36) and mammalian (Vero) cell cultures, and that it has a cryoprotectant capacity. Supplementation of cell culture media with sericin at 80 µg/mL resulted in a significant increase of 1 log in the concentration of ZIKV infectious particles produced from both cell lines. Furthermore, final virus yields increased between 5 and 10-fold in Vero cells and between 7 and 23-fold in C6/36 cells when sericin was supplemented, compared to control conditions. These results show that sericin is an effective supplement to increase ZIKV production by Vero and C6/36 cells. Additionally, sericin was a suitable cryoprotective agent, and hence an alternative to FBS and DMSO, for the cryopreservation of C6/36 cells but not for Vero cells.
Assuntos
Sericinas , Infecção por Zika virus , Zika virus , Animais , Técnicas de Cultura de Células/métodos , Chlorocebus aethiops , Humanos , Insetos , Mamíferos , Sericinas/metabolismo , Sericinas/farmacologia , Seda/metabolismo , Células Vero , Infecção por Zika virus/tratamento farmacológicoRESUMO
The dengue virus NS1 is a multifunctional protein that forms part of replication complexes. NS1 is also secreted, as a hexamer, to the extracellular milieu. Circulating NS1 has been associated with dengue pathogenesis by several mechanisms. Cell binding and internalization of soluble NS1 result in endothelial hyperpermeability and in the downregulation of the innate immune response. In this work, we report that the HDL scavenger receptor B1 (SRB1) in human hepatic cells and a scavenger receptor B1-like in mosquito C6/36 cells act as cell surface binding receptors for dengue virus NS1. The presence of the SRB1 on the plasma membrane of C6/36 cells, as well as in Huh7 cells, was demonstrated by confocal microscopy. The internalization of NS1 can be efficiently blocked by anti-SRB1 antibodies, and previous incubation of the cells with HDL significantly reduces NS1 internalization. Significant reduction in NS1 internalization was observed in C6/36 cells transfected with siRNAs specific for SRB1. In addition, the transient expression of SRB1 in Vero cells, which lacks the receptor, allows NS1 internalization in these cells. Direct interaction between soluble NS1 and the SRB1 in Huh7 and C6/36 cells was demonstrated in situ by proximity ligation assays and in vitro by surface plasmon resonance. Finally, results are presented indicating that the SRB1 also acts as a cell receptor for Zika virus NS1. These results demonstrate that dengue virus NS1, a bona fide lipoprotein, usurps the HDL receptor for cell entry and offers explanations for the altered serum lipoprotein homeostasis observed in dengue patients. IMPORTANCE Dengue is the most common viral disease transmitted to humans by mosquitoes. The dengue virus NS1 is a multifunctional glycoprotein necessary for viral replication. NS1 is also secreted as a hexameric lipoprotein and circulates in high concentrations in the sera of patients. Circulating NS1 has been associated with dengue pathogenesis by several mechanisms, including favoring of virus replication in hepatocytes and dendritic cells and disruption of the endothelial glycocalyx leading to hyperpermeability. Those last actions require NS1 internalization. Here, we identify the scavenger cell receptor B1, as the cell-binding receptor for dengue and Zika virus NS1, in cultured liver and in mosquito cells. The results indicate that flavivirus NS1, a bona fide lipoprotein, usurps the human HDL receptor and may offer explanations for the alterations in serum lipoprotein homeostasis observed in dengue patients.
Assuntos
Vírus da Dengue , Receptores Depuradores , Proteínas não Estruturais Virais , Infecção por Zika virus , Zika virus , Animais , Linhagem Celular , Chlorocebus aethiops , Culicidae/virologia , Dengue/virologia , Vírus da Dengue/metabolismo , Humanos , Lipoproteínas HDL , Receptores de Lipoproteínas , Receptores Depuradores/metabolismo , Células Vero , Proteínas não Estruturais Virais/imunologia , Internalização do Vírus , Zika virus/metabolismoRESUMO
OBJECTIVE: To develop a method for the efficient assembly of viral or multimeric proteins into virus-like particles (VLP) or other macro structures. RESULTS: Protein monomers were assembled by eliminating calcium ions through precipitation. The model protein, rotavirus VP6, assembled into stable, long nanotubes with better quality than the assemblies obtained directly from cell culture. Nanotube length was directly proportional to the initial concentration of VP6 monomers, in accordance with the classic nucleation theory of capsid assembly. The quality of the obtained assemblies was confirmed when the nanotubes were functionalized with metals, yielding unique nanobiomaterials. Assembly efficiency was improved in comparison with other previously proposed methods. CONCLUSIONS: The novel method presented here is simpler and faster than other reported methods for the assembly and disassembly of viral proteins, a step needed for most applications.
Assuntos
Antígenos Virais/química , Antígenos Virais/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Rotavirus/metabolismo , Cálcio/química , Precipitação Química , Nanotubos/química , Multimerização ProteicaRESUMO
The insect cell-baculovirus expression system technology (BEST) has a prominent role in producing recombinant proteins to be used as research and diagnostic reagents and vaccines. The glycosylation profile of proteins produced by the BEST is composed predominantly of terminal mannose glycans, and, in Trichoplusia ni cell lines, core α3 fucosylation, a profile different to that in mammals. Insects contain all the enzymatic activities needed for complex N- and O-glycosylation and sialylation, although few reports of complex glycosylation and sialylation by the BEST exist. The insect cell line and culture conditions determine the glycosylation profile of proteins produced by the BEST. The promoter used, dissolved oxygen tension, presence of sugar precursors, bovine serum or hemolymph, temperature, and the time of harvest all influence glycosylation, although more research is needed. The lack of activity of glycosylation enzymes possibly results from the transcription regulation and stress imposed by baculovirus infection. To solve this limitation, the glycosylation pathway of insect cells has been engineered to produce complex sialylated glycans and to eliminate α3 fucosylation, either by generating transgenic cell lines or by using baculovirus vectors. These strategies have been successful. Complex glycosylation, sialylation, and inhibition of α3 fucosylation have been achieved, although the majority of glycans still have terminal mannose residues. The implication of insect glycosylation in the proteins produced by the BEST is discussed. Graphical Abstract.
Assuntos
Baculoviridae , Insetos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Bovinos , Glicosilação , Insetos/metabolismo , Proteínas Recombinantes/genética , TecnologiaRESUMO
BACKGROUND: Chinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the great importance of precipitation methods for the processing of samples in proteomics, the acetone, methanol-chloroform (M/C), and trichloroacetic acid (TCA)-acetone protocols were compared for CHO cells in terms of protein recovery, band pattern resolution, and presence on SDS-PAGE. RESULTS: Higher recovery and similar band profile with cellular homogenates were obtained using acetone precipitation with ultrasonic bath cycles (104.18 ± 2.67%) or NaOH addition (103.12 ± 5.74%), compared to the other two protocols tested. TCA-acetone precipitates were difficult to solubilize, which negatively influenced recovery percentage (77.91 ± 8.79%) and band presence. M/C with ultrasonic homogenization showed an intermediate recovery between the other two protocols (94.22 ± 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MW proteins (< 15â¯kDa). CONCLUSIONS: These results help in the processing of samples of CHO cells for their proteomic study by means of an easily accessible, fast protocol, with an almost complete recovery of cellular proteins and the capture of the original complexity of the cellular composition. Acetone protocol could be incorporated to sample-preparation workflows in a straightforward manner and can probably be applied to other mammalian cell lines as well.
Assuntos
Animais , Proteínas Recombinantes , Células CHO , Proteômica/métodos , Acetona , Precipitação Química , Solubilidade , Ácido Tricloroacético , Separação Celular , Clorofórmio , Técnicas de Cultura de Células , Metanol , Eletroforese em Gel de PoliacrilamidaRESUMO
Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30-60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10-60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10-60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.
Assuntos
Microssomos/metabolismo , Proteômica , Animais , Células CHO , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Centrifugação , Cricetinae , Cricetulus , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Ontologia Genética , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Microssomos/ultraestrutura , Mitocôndrias/ultraestrutura , Proteoma/metabolismo , Software , Frações Subcelulares/metabolismoRESUMO
Chinese hamster ovary (CHO) cells are characterized by a low glucose catabolic efficiency, resulting in undesirable lactate production. Here, it is hypothesized that such low efficiency is determined by the transport of pyruvate into the mitochondria. The mitochondrial pyruvate carrier (MPC), responsible for introducing pyruvate into the mitochondria, is formed by two subunits, MPC1 and MPC2. Stable CHO cell lines, overexpressing the genes of both subunits, were constructed to facilitate the entry of pyruvate into the mitochondria and its incorporation into oxidative pathways. Significant overexpression of both genes, compared to the basal level of the control cells, was verified, and subcellular localization of both subunits in the mitochondria was confirmed. Kinetic evaluation of the best MPC overexpressing CHO cells showed a reduction of up to 50% in the overall yield of lactate production with respect to the control. An increase in specific growth rate and maximum viable cell concentration, as well as an increase of up to 40% on the maximum concentration of two recombinant model proteins transiently expressed (alkaline phosphatase or a monoclonal antibody), was also observed. Hybrid cybernetic modeling, that considered 89 reactions, 25 extracellular metabolites, and a network of 62 intracellular metabolites, explained that the best MPC overexpression case resulted in an increased metabolic flux across the mitochondrial membrane, activated a more balanced growth, and reduced the Warburg effect without compromising glucose consumption rate and maximum cell concentration. Overall, this study showed that transport of pyruvate into the mitochondria limits the efficiency of glucose oxidation, which can be overcome by a cell engineering approach.
Assuntos
Ácido Láctico/metabolismo , Engenharia Metabólica/métodos , Proteínas Mitocondriais , Transportadores de Ácidos Monocarboxílicos , Proteínas Recombinantes , Animais , Células CHO , Cricetinae , Cricetulus , Glucose/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Chinese hamster ovary (CHO) cell culture has a major importance on the production of biopharmaceuticals, including recombinant therapeutic proteins such as monoclonal antibodies (MAb). Mathematical modeling of biological systems can successfully assess metabolism complexity while providing logical and systematic methods for relevant genetic target and culture parameter identification toward cell growth and productivity improvements. Most modeling approaches on CHO cells have been performed under stationary constraints, and only a few dynamic models have been presented on simplified reaction sets, due to substantial overparameterization problems. The hybrid cybernetic modeling (HCM) approach has been recently used to describe the dynamic behavior by incorporating regulation between different metabolic states by elementary mode participation control, with sets of equations evaluated by objective functions. However, as metabolic networks evaluated are constructed toward a genomic scale, and cell compartmentalization is considered, identification of the active set becomes more difficult as EM number exponentially grows. Thus, the development of robust approaches for EM active set selection and analysis with smaller computational requirements is required to impulse the use of cybernetic modeling on larger up to genome-scale networks. In this report, a novel elementary mode selection strategy, based on a polar representation of the convex solution space is presented and coupled to a cybernetic approach to model the dynamic physiologic and metabolic behavior of CHO-S cell cultures. The proposed Polar Space Yield Analysis (PSYA) was compared to other reported elementary mode selection approaches derived from Common Metabolic Objective Analysis (CMOA) used in Flux Balance Analysis (FBA), Yield Space Analysis (YSA), and Lumped Yield Space Analysis (LYSA). For this purpose, exponential growth phase dynamic metabolic models were calculated using kinetic rate equations based on previously modeled growth parameters. Finally, complete culture dynamic metabolic flux models were constructed using the HCM approach with selected elementary mode sets. The yield space elementary mode- and the polar space elementary mode- hybrid cybernetic models presented the best fits and performances. Also, a flux reaction perturbation prediction approach based on the polar yield solution space resulted useful for metabolic network flux distribution capability analysis and identification of potential genetic modifications targets.
RESUMO
Biomedical applications increasingly require fully characterized new nanomaterials. There is strong evidence showing that nanomaterials not only interact with cells passively but also actively, mediating essential molecular processes for the regulation of cellular functions, but we are only starting to understand the mechanisms of those interactions. Systematic studies about cell behavior as a response to specific nanoparticle properties are scarce in the literature even when they are necessary for the rational design of medical nanodevices. Information in the literature shows that the physicochemical properties determine the bioactivity, biocompatibility, and safety of nanomaterials. The information available regarding the interaction and responses of cells to nanomaterials has not been analyzed and discussed in a single document. Hence, in this review, we present the latest advances about cellular responses to nanomaterials and integrate the available information into concrete considerations for the development of innovative, efficient, specific and, more importantly, safe biomedical nanodevices. We focus on how physicochemical nanoparticle properties (size, chemical surface, shape, charge, and topography) influence cell behavior in a first attempt to provide a practical guide for designing medical nanodevices, avoiding common experimental omissions that may lead to data misinterpretation. Finally, we emphasize the importance of the systematic study of nano-bio interactions to acquire sufficient reproducible information that allows accurate control of cell behavior based on tuning of nanomaterial properties. This information is useful to guide the design of specific nanodevices and nanomaterials to elicit desired cell responses, like targeting, drug delivery, cell attachment, differentiation, etc, or to avoid undesired side effects.
Assuntos
Desenho de Equipamento/métodos , Nanoestruturas/química , Animais , Comunicação Celular , Sistemas de Liberação de Medicamentos , Humanos , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
BACKGROUND: Simultaneous co-fermentation of mixed sugars is an important feature to consider in the production of ethanol from lignocellulosic biomass hydrolysates because it enhances the overall ethanol yield and volumetric productivity during fermentation. Continuous cultures can be used during ethanol production from lignocellulosic hydrolysates to prevent catabolite repression by glucose on other sugars, such as xylose, and thus promote the simultaneous and total consumption of sugars and reduce fermentation time. The use of single- and two-stage continuous cultures under micro-aerated conditions for simultaneous consumption of xylose and glucose, and fermentation to ethanol by ethanologenic Escherichia coli strain MS04 was studied. Mineral medium supplemented with glucose, xylose and sodium acetate, was used to compare continuous cultures performance to batch cultures. RESULTS: Single-stage continuous cultures under micro-aerated conditions allowed the total co-consumption of a mixture of glucose and xylose (7.5 and 42.5 g/L, respectively) in mineral medium, with steady state ethanol production of 18 g/L, and a volumetric ethanol productivity of 0.9 g/L h, when low dilution rates (0.05 h-1) were used. However, the volumetric productivity was lower than the batch process under similar conditions (1.3 g/L h). Conversely, micro-aerated two-stage continuous culture enhanced the volumetric productivity up to 1.6 g/L h at a dilution rate of 0.15 h-1, with a total consumption of sugars and a slight reduction of the overall ethanol yield. CONCLUSIONS: The total and simultaneous consumption of glucose and xylose by the ethanologenic E. coli strain MS04 was accomplished by using two-stage continuous culture under micro-aerated conditions with an increase in the volumetric ethanol productivity of 23% and 78% when compared to batch and single-stage continuous cultures, respectively. Multi-stage continuous cultivation can be used to promote the simultaneous consumption of all sugars contained in biomass hydrolysates, and thus increase the volumetric ethanol productivity of the fermentation process.
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Técnicas de Cultura Celular por Lotes , Dissacarídeos/metabolismo , Escherichia coli/metabolismo , Etanol/metabolismo , FermentaçãoRESUMO
The Ebola virus disease is a public health challenge. To date, the only available treatments are medical support or the emergency administration of experimental drugs. The absence of licensed vaccines against Ebola virus impedes the prevention of infection. Vaccines based on recombinant virus-like particles (VLP) are a promising alternative. The Zaire Ebola virus serotype (ZEBOV) is the most aggressive with the highest mortality rates. Production of ZEBOV-VLP has been accomplished in mammalian and insect cells by the recombinant coexpression of three structural proteins, the glycoprotein (GP), the matrix structural protein VP40, and the nucleocapsid protein (NP). However, specific conditions to manipulate protein concentrations and improve assembly into VLP have not been determined to date. Here, we used a design of experiments (DoE) approach to determine the best MOI and TOI for three recombinant baculoviruses: bac-GP, bac-VP40 and bac-NP, each coding for one of the main structural proteins of ZEBOV. We identified two conditions where the simultaneous expression of the three recombinant proteins was observed. Interestingly, a temporal and stoichiometric interplay between the three structural proteins was observed. VP40 was required for the correct assembly of ZEBOV-VLP. High NP concentrations reduced the accumulation of GP, which has been reported to be necessary for inducing a protective immune response. Electron microscopy showed that the ZEBOV-VLP produced were morphologically similar to the native virus micrographs previously reported in the literature. A strategy for producing ZEBOV in insect cells, which consists in using a high MOI of bac-VP40 and bac-GP, and reducing expression of NP, either by delaying infection or reducing the MOI of bac-NP, was the most adequate for the production of VLP.
Assuntos
Baculoviridae/imunologia , Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Insetos/imunologia , Insetos/virologia , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/imunologia , Proteínas do Nucleocapsídeo/imunologia , Nucleoproteínas/imunologia , Células Sf9 , Proteínas do Core Viral/imunologia , Proteínas da Matriz Viral/imunologiaRESUMO
BACKGROUND: Classic metabolic engineering strategies often induce significant flux imbalances to microbial metabolism, causing undesirable outcomes such as suboptimal conversion of substrates to products. Several mathematical frameworks have been developed to understand the physiological and metabolic state of production strains and to identify genetic modification targets for improved bioproduct formation. In this work, a modeling approach was applied to describe the physiological behavior and the metabolic fluxes of a shikimic acid overproducing Escherichia coli strain lacking the major glucose transport system, grown on complex media. RESULTS: The obtained flux distributions indicate the presence of high fluxes through the pentose phosphate and Entner-Doudoroff pathways, which could limit the availability of erythrose-4-phosphate for shikimic acid production even with high flux redirection through the pentose phosphate pathway. In addition, highly active glyoxylate shunt fluxes and a pyruvate/acetate cycle are indicators of overflow glycolytic metabolism in the tested conditions. The analysis of the combined physiological and flux response surfaces, enabled zone allocation for different physiological outputs within variant substrate conditions. This information was then used for an improved fed-batch process designed to preserve the metabolic conditions that were found to enhance shikimic acid productivity. This resulted in a 40% increase in the shikimic acid titer (60 g/L) and 70% increase in volumetric productivity (2.45 gSA/L*h), while preserving yields, compared to the batch process. CONCLUSIONS: The combination of dynamic metabolic modeling and experimental parameter response surfaces was a successful approach to understand and predict the behavior of a shikimic acid producing strain under variable substrate concentrations. Response surfaces were useful for allocating different physiological behavior zones with different preferential product outcomes. Both model sets provided information that could be applied to enhance shikimic acid production on an engineered shikimic acid overproducing Escherichia coli strain.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Biológicos , Ácido Chiquímico/metabolismo , Transporte Biológico/genética , Carbono/metabolismo , Escherichia coli/crescimento & desenvolvimento , Glucose/metabolismo , Engenharia Metabólica , Análise do Fluxo MetabólicoRESUMO
Heterogeneity of cellular populations has been frequently observed. We used live cell imaging to follow Sf9 insect cells before and after infection with baculovirus, to understand population dynamics. It was possible to identify in real time cells with distinctive phenotypes. Mobile cells with an elongated bipolar shape were observed. They extended pseudopods and actively moved about the culture surface. The presence of actively moving elongated cells increased when cultures were subjected to oxygen limiting or excessive conditions, suggesting that stress triggered differentiation of cells to the mobile phenotype. A dual reporter baculovirus (DRBac), coding for two fluorescent proteins under promoters with different temporality, was designed to follow sequential phenomena through infection. Oxygen limitation reduced the number of cells that expressed the reporter proteins, possibly because it reduced the efficiency of baculovirus infection. Elongated cells did not show signs of infection. To our knowledge, this is the first time that actively moving cells are observed in real time in Sf9 cultures, which had distinctive responses towards infection. Anoxia was identified as a factor that modulates baculovirus infection. Results open a new approach for understanding the insect-cell baculovirus system. Particular cellular phenotypes with unique traits can be isolated for specific applications.
Assuntos
Técnicas de Cultura de Células/métodos , Microscopia de Vídeo/métodos , Análise de Célula Única/métodos , Animais , Baculoviridae , Proliferação de Células , Forma Celular , Corantes Fluorescentes/química , Microscopia de Fluorescência , Fagocitose , Células Sf9RESUMO
Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28-34°C is an effective strategy to expand the productive life period of cells and increase their productivity (qp). Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system.