RESUMO
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.
Assuntos
Vias Biossintéticas , Peptídeos , Cricetinae , Animais , Cricetulus , Proteólise , Células CHO , Proteínas RecombinantesRESUMO
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
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
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
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
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
Dengue virus nonstructural protein 1 (NS1) is a multifunctional glycoprotein. For decades, the notion in the field was that NS1 is secreted exclusively from vertebrate cells and not from mosquito cells. However, recent evidence shows that mosquito cells also secrete NS1 efficiently. In this review, we discuss the evidence for secretion of NS1 of dengue virus, and of other flaviviruses, from mosquito cells, differences between NS1 secreted from mosquito and NS1 secreted from vertebrate cells, and possible roles of soluble NS1 in the insect flavivirus vector.
Assuntos
Culicidae/virologia , Vírus da Dengue/fisiologia , Dengue/virologia , Mosquitos Vetores , Proteínas não Estruturais Virais/metabolismo , Animais , HumanosRESUMO
INTRODUCTION AND AIM: Hepatic encephalopathy (HE), caused by hyperammonemia resulting from liver disease, is a spectrum of neuropsychiatric and motor disorders that can lead to death. Existing therapies are deficient and alternative treatments are needed. We have shown that gene therapy with a baculovirus vector containing the glutamine synthetase (Bac-GS) gene is efficient for reducing ammonia levels in an acute hyperammonemia rat model. However, the most common condition resulting from liver disease is chronic hyperammonemia. In this work, Bac-GS was evaluated in bile-duct ligated rats, a chronic liver disease model with hyperammonemia and some characteristics of Type C HE. MATERIAL AND METHODS: Bac-GS was tested for mediating GS overexpression in HeLa cells and H9C2 myotubes. For determining the utility of Bac-GS for the reduction of ammonia levels in a chronic hyperammonemia animal model, four groups of rats were treated: control, sham, ligated with Bac-GS and ligated with Bac-GFP. Baculoviruses were injected i.m. 18 days post-surgery. Blood was drawn 2, 3 and 4 weeks post-surgery and plasma ammonia concentrations were quantified. RESULTS: In protein lysates of cells and myotubes transduced with Bac-GS, a 44 kDa band corresponding to GS was detected. Significant results were obtained in the hyperammonemic bile-duct ligated rat model, as plasma ammonia was reduced to normal levels 3 days after treatment with Bac-GS. Furthermore, a transitory effect of Bac-GS was observed. CONCLUSION: Our results show that gene therapy by delivering GS is a promising alternative for treatment of hyperammonemia in acute-on-chronic liver failure patients with HE.
Assuntos
Baculoviridae/genética , Terapia Genética/métodos , Encefalopatia Hepática/etiologia , Encefalopatia Hepática/terapia , Hiperamonemia/complicações , Análise de Variância , Animais , Western Blotting , Células Cultivadas , Doença Crônica , Modelos Animais de Doenças , Vetores Genéticos , Glutamato-Amônia Ligase/administração & dosagem , Células HeLa/citologia , Células HeLa/patologia , Encefalopatia Hepática/patologia , Humanos , Hiperamonemia/fisiopatologia , Distribuição Aleatória , Ratos , Fatores de Risco , Sensibilidade e EspecificidadeRESUMO
Dissolved carbon dioxide (dCO2 ) accumulation during cell culture has been recognized as an important parameter that needs to be controlled for successful scale-up of animal cell culture because above a certain concentration there are adverse effects on cell growth performance and protein production. We investigated the effect of accumulation of dCO2 in bioreactor cultures of expresSF+(®) insect cells infected with recombinant baculoviruses expressing recombinant influenza virus hemagglutinins (rHA). Different strategies for bioreactor cultures were used to obtain various ranges of concentrations of dCO2 (<50, 50-100, 100-200, and >200 mmHg) and to determine their effects on recombinant protein production and cell metabolic activity. We show that the accumulation of dCO2 at levels > 100 mmHg resulted in reduced metabolic activity, slowed cell growth, prolonged culture viability after infection, and decreased infection kinetics. The reduced rHA yields were not caused by the decrease in the extracellular pH that resulted from dCO2 accumulation, but were most likely due to the effect of dCO2 accumulation in cells. The results obtained here at the 2 L scale have been used for the design of large-scale processes to manufacture the rHA based recombinant vaccine Flublok™ at the 2500 L scale Biotechnol. Bioeng. 2015;112: 2267-2275. © 2015 Wiley Periodicals, Inc.
Assuntos
Dióxido de Carbono/análise , Meios de Cultura/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vacinas contra Influenza/metabolismo , Animais , Reatores Biológicos , Linhagem Celular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Concentração de Íons de Hidrogênio , Vacinas contra Influenza/genética , Insetos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vacinas Sintéticas/genética , Vacinas Sintéticas/metabolismoRESUMO
Live oral rotavirus (RV) vaccines are part of routine childhood immunization but are associated with adverse effects, particularly intussusception. We have developed a non-live combined RV - norovirus (NoV) vaccine candidate consisting of human RV inner-capsid rVP6 protein and NoV virus-like particles. To determine the effect of delivery route on induction of VP6-specific protective immunity, BALB/c mice were administered a vaccine containing RV rVP6 intramuscularly, intranasally or a combination of both, and challenged with murine RV. At least 65 % protection against RV shedding was observed regardless of delivery route. The levels of post-challenge serum VP6-specific IgA titers correlated with protection.
Assuntos
Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Infecções por Rotavirus/prevenção & controle , Vacinas contra Rotavirus/imunologia , Rotavirus/imunologia , Administração Intranasal , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/administração & dosagem , Antígenos Virais/genética , Proteínas do Capsídeo/administração & dosagem , Proteínas do Capsídeo/genética , Feminino , Humanos , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Rotavirus/genética , Infecções por Rotavirus/imunologia , Infecções por Rotavirus/virologia , Vacinas contra Rotavirus/administração & dosagem , Vacinas contra Rotavirus/genéticaRESUMO
Endogenous glycosylated Hev b 2 (endo-ß-1,3-glucanase) from Hevea brasiliensis is an important latex allergen that is recognized by IgE antibodies from patients who suffer from latex allergy. The carbohydrate moieties of Hev b 2 constitute a potentially important IgE-binding epitope that could be responsible for its cross-reactivity. Here, the structure of the endogenous isoform II of Hev b 2 that exhibits three post-translational modifications, including an N-terminal pyroglutamate and two glycosylation sites at Asn27 and at Asn314, is reported from two crystal polymorphs. These modifications form a patch on the surface of the molecule that is proposed to be one of the binding sites for IgE. A structure is also proposed for the most important N-glycan present in this protein as determined by digestion with specific enzymes. To analyze the role of the carbohydrate moieties in IgE antibody binding and in human basophil activation, the glycoallergen was enzymatically deglycosylated and evaluated. Time-lapse automated video microscopy of basophils stimulated with glycosylated Hev b 2 revealed basophil activation and degranulation. Immunological studies suggested that carbohydrates on Hev b 2 represent an allergenic IgE epitope. In addition, a dimer was found in each asymmetric unit that may reflect a regulatory mechanism of this plant defence protein.
Assuntos
Antígenos de Plantas/química , Basófilos/efeitos dos fármacos , Celulase/química , Hevea/química , Imunoglobulina E/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Antígenos de Plantas/imunologia , Antígenos de Plantas/isolamento & purificação , Antígenos de Plantas/farmacologia , Teste de Degranulação de Basófilos , Basófilos/citologia , Basófilos/imunologia , Sítios de Ligação , Sequência de Carboidratos , Células Cultivadas , Celulase/imunologia , Celulase/isolamento & purificação , Celulase/farmacologia , Cristalografia por Raios X , Ácido Glutâmico/metabolismo , Glicosilação , Humanos , Imunoglobulina E/imunologia , Hipersensibilidade ao Látex/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/imunologia , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Imagem com Lapso de TempoRESUMO
Biomolecules are advantageous scaffolds for the synthesis and ordering of metallic nanoparticles. Rotavirus VP6 nanotubes possess intrinsic affinity to metal ions, a property that has been exploited to synthesize gold nanoparticles over them. The resulting nanobiomaterials have unique properties useful for novel applications. However, the formed nanobiomaterials lack of colloidal stability and flocculate, limiting their functionality. Here we demonstrate that it is possible to synthesize thiol-protected gold nanoparticles over VP6 nanotubes, which resulted in soluble nanobiomaterials. With this strategy, it was possible to modulate the size, colloidal stability, and surface plasmon resonance of the synthesized nanoparticles by controlling the content of the thiolated ligands. Two types of water-soluble ligands were tested, a small linear ligand, sodium 3-mercapto-1-propanesulfonate (MPS), and a bulky ligand, 5-mercaptopentyl ß-D-glucopyranoside (GlcC5SH). The synthesized nanobiomaterials had a higher stability in suspension, as determined by Z-potential measurements. To the extent of our knowledge, this is the first time that a rational strategy is developed to modulate the particular properties of metal nanoparticles in situ synthesized over a protein bioscaffold through thiol coating, achieving a high spatial and structural organization of nanoparticles in a single integrative hybrid structure.
Assuntos
Antígenos Virais/química , Proteínas do Capsídeo/química , Ouro/química , Nanotubos/química , Compostos de Sulfidrila/química , Complexos de Coordenação/química , Tamanho da PartículaRESUMO
BACKGROUND: Protein assemblies, such as virus-like particles, have increasing importance as vaccines, delivery vehicles and nanomaterials. However, their use requires stable assemblies. An important cause of loss of stability in proteins is oxidation, which can occur during their production, purification and storage. Despite its importance, very few studies have investigated the effect of oxidation in protein assemblies and their structural units. In this work, we investigated the role of in vitro oxidation in the assembly and stability of rotavirus VP6, a polymorphic protein. RESULTS: The susceptibility to oxidation of VP6 assembled into nanotubes (VP6NT) and unassembled VP6 (VP6U) was determined and compared to bovine serum albumin (BSA) as control. VP6 was more resistant to oxidation than BSA, as determined by measuring protein degradation and carbonyl content. It was found that assembly protected VP6 from in vitro metal-catalyzed oxidation. Oxidation provoked protein aggregation and VP6NT fragmentation, as evidenced by dynamic light scattering and transmission electron microscopy. Oxidative damage of VP6 correlated with a decrease of its center of fluorescence spectral mass. The in vitro assembly efficiency of VP6U into VP6NT decreased as the oxidant concentration increased. CONCLUSIONS: Oxidation caused carbonylation, quenching, and destruction of aromatic amino acids and aggregation of VP6 in its assembled and unassembled forms. Such modifications affected protein functionality, including its ability to assemble. That assembly protected VP6 from oxidation shows that exposure of susceptible amino acids to the solvent increases their damage, and therefore the protein surface area that is exposed to the solvent is determinant of its susceptibility to oxidation. The inability of oxidized VP6 to assemble into nanotubes highlights the importance of avoiding this modification during the production of proteins that self-assemble. This is the first time that the role of oxidation in protein assembly is studied, evidencing that oxidation should be minimized during the production process if VP6 nanotubes are required.
Assuntos
Antígenos Virais/química , Proteínas do Capsídeo/química , Metais/química , Rotavirus/fisiologia , Animais , Antígenos Virais/genética , Antígenos Virais/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Bovinos , Compostos Ferrosos/química , Peróxido de Hidrogênio/química , Luz , Nanotubos/química , Oxirredução , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espalhamento de Radiação , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Montagem de VírusRESUMO
Rotavirus VP6 nanotubes are an attractive option for a recombinant vaccine against rotavirus disease. Protection against rotavirus infection and an adjuvant effect have been observed upon immunization with VP6 nanotubes. However, little information exists on how VP6 nanotubes interact with cells and trigger an immune response. In this work, the interaction between VP6 nanotubes and different cell lines was characterized. VP6 nanotubes were not cytotoxic to any of the animal or human cell lines tested. Uptake of nanotubes into cells was cell-line-dependent, as only THP1 and J774 macrophage cells internalized them. Moreover, the size and spatial arrangement of VP6 assembled into nanotubes allowed their uptake by macrophages, as double-layered rotavirus-like particles also displaying VP6 in their surface were not taken up. The internalization of VP6 nanotubes was inhibited by methyl-ß-cyclodextrin, but not by genistein, indicating that nanotube entry is specific, depends on the presence of cholesterol in the plasma membrane, and does not require the activity of tyrosine kinases. The information generated here expands our understanding of the interaction of protein nanotubes with cells, which is useful for the application of VP6 nanotubes as a vaccine.
Assuntos
Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Nanotubos/virologia , Rotavirus/fisiologia , Vacinas Sintéticas , Internalização do Vírus , Animais , Células COS , Células CACO-2 , Chlorocebus aethiops , Colesterol , Endocitose/efeitos dos fármacos , Genisteína/farmacologia , Células HEK293 , Humanos , Nanotubos/química , Proteínas Tirosina Quinases , Rotavirus/imunologia , Vacinas Virais/imunologia , beta-Ciclodextrinas/farmacologiaRESUMO
New technologies require the development of novel nanomaterials that need to be fully characterized to achieve their potential. High-resolution low-voltage scanning transmission electron microscopy (STEM) has proven to be a very powerful technique in nanotechnology, but its use for the characterization of nanobiomaterials has been limited. Rotavirus VP6 self-assembles into nanotubular assemblies that possess an intrinsic affinity for Au ions. This property was exploited to produce hybrid nanobiomaterials by the in situ functionalization of recombinant VP6 nanotubes with gold nanoparticles. In this work, Raman spectroscopy and advanced analytical electron microscopy imaging with spherical aberration-corrected (Cs) STEM and nanodiffraction at low-voltage doses were employed to characterize nanobiomaterials. STEM imaging revealed the precise structure and arrangement of the protein templates, as well as the nanostructure and atomic arrangement of gold nanoparticles with high spatial sub-Angstrom resolution and avoided radiation damage. The imaging was coupled with backscattered electron imaging, ultra-high resolution scanning electron microscopy and x-ray spectroscopy. The hybrid nanobiomaterials that were obtained showed unique properties as bioelectronic conductive devices and showed enhanced Raman scattering by their precise arrangement into superlattices, displaying the utility of viral assemblies as functional integrative self-assembled nanomaterials for novel applications.
Assuntos
Antígenos Virais/química , Materiais Biocompatíveis/química , Proteínas do Capsídeo/química , Microscopia Eletrônica de Transmissão e Varredura , Nanotubos/química , Análise Espectral Raman , Ouro/química , Processamento de Imagem Assistida por Computador , Nanopartículas Metálicas/química , Proteínas Recombinantes/químicaRESUMO
Virus-like particles (VLPs) of the adeno-associated virus (AAV) can be produced using the baculovirus expression vector system. Insertion of small peptides on the surface of the AAV or AAV VLPs has been used to redirect the AAV to different target tissues and for vaccine development. Usually, the VLPs self-assemble intracellularly, and an extraction step must be performed before purification. Here, we describe the method we have used to extract AAV VLPs from insect cells successfully with peptide insertions on their surface.
Assuntos
Dependovirus , Peptídeos , Dependovirus/genética , Animais , Peptídeos/química , Peptídeos/genética , Vetores Genéticos/genética , Vírion/genética , Baculoviridae/genética , Células Sf9 , Humanos , Linhagem Celular , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/isolamento & purificaçãoRESUMO
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.
Assuntos
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).
Assuntos
Bromovirus , Bromovirus/genética , Animais , Baculoviridae/genética , Vetores Genéticos/genética , Cromatografia por Troca Iônica/métodos , Vírion/isolamento & purificação , Vírion/genética , Vírion/metabolismoRESUMO
Biological molecules that self-assemble in the nanoscale range are useful multifunctional materials. Rotavirus VP6 protein self-assembles into tubular structures in the absence of other rotavirus proteins. Here, we present strategies for selectively directing metal functionalization to the lumen of VP6 nanotubes. The specific in situ metal reduction in the inner surface of nanotube walls was achieved by the simple modification of a method previously reported to functionalize the nanotube outer surface. Silver nanorods and nanowires as long as 1.5 µm were formed inside the nanotubes by coalescence of nanoparticles. Such one-dimensional structures were longer than others previously obtained using bioscaffolds. The interactions between silver ions and the nanotube were simulated to understand the conditions that allowed nanowire formation. Molecular docking showed that a naturally occurring arrangement of aspartate residues enabled the stabilization of silver ions on the internal surface of the VP6 nanotubes. This is the first time that such a spatial arrangement has been proposed for the nucleation of silver nanoparticles, opening the possibility of using such an array to direct functionalization of other biomolecules. These results demonstrate the natural capabilities of VP6 nanotubes to function as a versatile biotemplate for nanomaterials.
Assuntos
Antígenos Virais/química , Proteínas do Capsídeo/química , Nanotubos/química , Nanofios/química , Prata/química , Antígenos Virais/ultraestrutura , Sítios de Ligação , Proteínas do Capsídeo/ultraestrutura , Materiais Revestidos Biocompatíveis , Ouro/química , Íons , Ligantes , Modelos Moleculares , Nanotubos/ultraestrutura , Nanofios/ultraestrutura , Paládio/químicaRESUMO
The insect cell-baculovirus system (IC-BEVS) is widely used for the production of recombinant viral proteins for vaccine applications. It is especially suitable for the production of virus-like particles, which often require the simultaneous production of several recombinant proteins. Here, the available tools and process requirements for the simultaneous production of several recombinant proteins using the IC-BEVS are discussed. The production of double-layered rotavirus like particles is used as a specific example for the simultaneous production of two recombinant proteins. Methods to quantify VLP in small samples are described. The multiplicity and time of infection are presented as tools to manipulate protein concentration, and the effect on protein concentration ratios on the assembly efficiency of double-layered rotavirus like particles is discussed. It was found that not only the ratio between the recombinant proteins is determinant of VLP assembly efficiency, but also that assembly efficiency is related to the characteristics of the assembled proteins. This is the first time that kinetics of VLP production are followed during cultures, and that the assembly efficiency is quantitatively determined.