RESUMO
BACKGROUND: Advantages of translocation of recombinant proteins to the periplasm in Escherichia coli include simplified downstream processing, and improved folding and in vivo activity of the target protein. There are, however, problems encountered in the periplasmic production that can be associated with the incorrect formation of disulfide bonds, incomplete cleavage of the signal peptide, and proteolytic degradation. A common strategy used to overcome these difficulties involves manipulating the cellular levels of proteases and periplasmic folding assistants like chaperones, signal peptide peptidases or thiol-disulfide oxidoreductases. To date, this has been achieved by plasmid-based over-expression or knockouts of the relevant genes. RESULTS: We changed the translation efficiencies of five native E. coli proteins, DsbA, DsbB, Skp, SppA, and DegP, by modifying the strength of their ribosome binding sites (RBS). The genomic RBS sequences were replaced with synthetic ones that provided a predicted translation initiation rate. Single- and double-gene mutant strains were created and tested for production of two pharmaceutically relevant proteins, PelB-scFv173-2-5-AP and OmpA-GM-CSF. Almost all the single-gene mutant strains showed improved periplasmic production of at least one of the recombinant proteins. No further positive effects were observed when the mutations were combined. CONCLUSIONS: Our findings confirm that our strain engineering approach involving translational regulation of endogenous proteins, in addition to plasmid-based methods, can be used to manipulate the cellular levels of periplasmic folding assistants and proteases to improve the yields of translocated recombinant proteins. The positive effects of SppA overexpression should be further investigated in E. coli.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Peptídeo Hidrolases/metabolismo , Periplasma/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Transporte Proteico , Proteínas Recombinantes/metabolismo , Proteínas de Bactérias , Proteínas de Ligação a DNA , Endopeptidases , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Edição de Genes , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Proteínas de Membrana , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Plasmídeos , Isomerases de Dissulfetos de Proteínas , Processamento de Proteína Pós-Traducional/genética , Proteínas Recombinantes/genéticaRESUMO
Human metapneumovirus (HMPV) may cause severe respiratory disease. The early innate immune response to viruses like HMPV is characterized by induction of antiviral interferons (IFNs) and proinflammatory immune mediators that are essential in shaping adaptive immune responses. Although innate immune responses to HMPV have been comprehensively studied in mice and murine immune cells, there is less information on these responses in human cells, comparing different cell types infected with the same HMPV strain. The aim of this study was to characterize the HMPV-induced mRNA expression of critical innate immune mediators in human primary cells relevant for airway disease. In particular, we determined type I versus type III IFN expression in human epithelial cells and monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs). In epithelial cells, HMPV induced only low levels of IFN-ß mRNA, while a robust mRNA expression of IFN-λs was found in epithelial cells, MDMs, and MDDCs. In addition, we determined induction of the interferon regulatory factors (IRFs) IRF1, IRF3, and IRF7 and critical inflammatory cytokines (IL-6, IP-10, and IL-1ß). Interestingly, IRF1 mRNA was predominantly induced in MDMs and MDDCs. Overall, our results suggest that for HMPV infection of MDDCs, MDMs, NECs, and A549 cells (the cell types examined), cell type is a strong determinator of the ability of HMPV to induce different innate immune mediators. HMPV induces the transcription of IFN-ß and IRF1 to higher extents in MDMs and MDDCs than in A549s and NECs, whereas the induction of type III IFN-λ and IRF7 is considerable in MDMs, MDDCs, and A549 epithelial cells.
Assuntos
Imunidade Inata/fisiologia , Metapneumovirus/patogenicidade , Infecções por Paramyxoviridae/imunologia , Células A549 , Células Cultivadas , Quimiocina CXCL10/metabolismo , Imunofluorescência , Humanos , Imunidade Inata/genética , Interleucina-6/metabolismo , Macrófagos/metabolismo , Metapneumovirus/imunologia , Microscopia Confocal , Infecções por Paramyxoviridae/metabolismo , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
Candida antarctica lipase B (CalB) was efficiently expressed (6.2 g L-1) in Escherichia coli by utilizing an N-terminal tag cassette and the XylS/Pm expression system in a fed-batch bioreactor; subsequent direct binding to EziG from crude extracts resulted in an immobilized catalyst with superior activity to Novozym 435.
RESUMO
Human metapneumovirus (hMPV) causes severe airway infection in children that may be caused by an unfavorable immune response. The nature of the innate immune response to hMPV in naturally occurring infections in children is largely undescribed, and it is unknown if inflammasome activation is implicated in disease pathogenesis. We examined nasopharynx aspirates and blood samples from hMPV-infected children without detectable co-infections. The expression of inflammatory and antiviral genes were measured in nasal airway secretions by relative mRNA quantification while blood plasma proteins were determined by a multiplex immunoassay. Several genes were significantly up-regulated at mRNA and protein level in the hMPV infected children. Most apparent was the expression of the chemokine IP-10, the pro-inflammatory cytokine IL-18 in addition to the interferon inducible gene ISG54. Interestingly, children experiencing more severe disease, as indicated by a severity index, had significantly more often up-regulation of the inflammasome-associated genes IL-1ß and NLRP3. Overall, our data point to cytokines, particularly inflammasome-associated, that might be important in hMPV mediated lung disease and the antiviral response in children with severe infection. Our study is the first to demonstrate that inflammasome components are associated with increased illness severity in hMPV-infected children.
Assuntos
Antivirais/uso terapêutico , Citocinas/genética , Metapneumovirus/patogenicidade , Infecções por Paramyxoviridae/genética , Infecções por Paramyxoviridae/virologia , Antivirais/farmacologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Humanos , Lactente , Inflamassomos/genética , Inflamassomos/metabolismo , Interferons/genética , Interferons/metabolismo , Masculino , Metapneumovirus/efeitos dos fármacos , Nasofaringe/patologia , Nasofaringe/virologia , Infecções por Paramyxoviridae/sangue , Infecções por Paramyxoviridae/tratamento farmacológicoRESUMO
The blood-brain barrier (BBB), composed of tightly organized endothelial cells, limits the availability of drugs to therapeutic targets in the central nervous system. The barrier is maintained by membrane bound efflux pumps efficiently transporting specific xenobiotics back into the blood. The efflux pump P-glycoprotein (P-gp), expressed at high levels in brain endothelial cells, has several drug substrates. Consequently, siRNA mediated silencing of the P-gp gene is one possible strategy how to improve the delivery of drugs to the brain. Herein, we investigated the potential of siRNA-chitosan nanoparticles in silencing P-gp in a BBB model. We show that the transfection of rat brain endothelial cells mediated effective knockdown of P-gp with subsequent decrease in P-gp substrate efflux. This resulted in increased cellular delivery and efficacy of the model drug doxorubicin.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Quitosana/química , Portadores de Fármacos/química , Células Endoteliais/efeitos dos fármacos , Nanopartículas/química , RNA Interferente Pequeno/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Transporte Biológico , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Doxorrubicina/farmacologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Cinética , Ratos , TransfecçãoRESUMO
The success of gene therapy depends on efficient delivery of DNA and requires a vector. A promising non-viral vector is chitosan. We tailored chitosan to optimize it for transfection by synthesizing self-branched and trisaccharide-substituted chitosan oligomers (SBTCO), which show superior transfection efficacy compared with linear chitosan (LCO). The aim of the work was to compare the cellular uptake and endocytic pathways of polyplexes formed by LCO and SBTCO. Both polyplexes were taken up by the majority of the cells, but the uptake of LCO was lower than SBTCO polyplexes. LCO polyplexes were internalized through both clathrin-dependent and clathrin-independent pathways, whereas SBTCO polyplexes were primarily taken up by clathrin-independent endocytosis. The different level of cellular uptake and the distinct endocytic pathways, may explain the difference in transfection efficacy. This was supported by the observation that photochemical internalization increased the transfection by LCO polyplexes considerably, whereas no effect on transfection was found for SBTCO polyplexes.
Assuntos
Cavéolas/metabolismo , Quitosana/química , Quitosana/metabolismo , Clatrina/metabolismo , DNA/metabolismo , Endocitose , Nanopartículas , Cavéolas/efeitos dos fármacos , Clorpromazina/farmacologia , DNA/genética , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Endocitose/efeitos dos fármacos , Genisteína/farmacologia , Células HeLa , Humanos , Hidrazonas/farmacologia , Propriedades de Superfície , Temperatura , TransfecçãoRESUMO
Chitosan has gained increasing interest for siRNA delivery. Although chitosan covers a family of structurally different polysaccharides, most siRNA delivery studies have been performed with conventional partially N-acetylated chitosans. Herein, the purpose was to identify fundamental chitosan molecular properties favoring siRNA delivery and efficient gene silencing in mammalian cells. Nanoparticles were prepared from well-defined chitosans of various chemical compositions, degrees of polymerization (DP(n)) and chain architectures. Structure-activity relationships were determined by the cellular uptake of siRNA and the knockdown efficiency at mRNA and protein levels. Additionally, the nanoparticle cytotoxicity was evaluated on the basis of cellular metabolic activity and membrane integrity. Our results show that the most efficient gene silencing was achieved using fully de-N-acetylated chitosans with intermediate chain lengths (DP(n) 100-300). These chitosans mediated efficient siRNA delivery at low siRNA concentrations and, in several cell lines, potent long-term silencing of both exogenous and endogenous target genes, with minimal cytotoxicity.
Assuntos
Quitosana/administração & dosagem , Portadores de Fármacos/administração & dosagem , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inativação Gênica , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/genética , Proteínas de Fluorescência Verde/genética , Humanos , Transfecção/métodosRESUMO
Chitosan possesses many characteristics of an ideal gene delivery system. However, the transfection efficiency of conventional chitosans is generally found to be low. In this study, we investigated the self-branching of chitosans as a strategy to improve its gene transfer properties without compromising its safety profile. Self-branched (SB) and self-branched trisaccharide-substituted (SBTCO) chitosans with molecular weights of 11-71 kDa were synthesized, characterized, and compared with their linear counterparts with respect to transfection efficiency, cellular uptake, formulation stability, and cytotoxicity. Our studies show that in contrast with unmodified linear chitosans that were unable to transfect HeLa cells, self-branched chitosans mediated high transfection efficiencies. The most efficient chitosan, SBTCO30, yielded gene expression levels two and five times higher than those of Lipofectamine and Exgen, respectively, and was nontoxic to cells. Nanoparticles formed with SBTCO chitosans exhibited a higher colloidal stability of formulation, efficient internalization without excessive cell surface binding, and low cytotoxicity.