RESUMO
The baculovirus expression vector system (BEVS) is recognized as a powerful platform for producing challenging proteins and multiprotein complexes both in academia and industry. Since a baculovirus was first used to produce heterologous human IFN-ß protein in insect cells, the BEVS has continuously been developed and its applications expanded. We have recently established a multigene expression toolbox (HR-bac) composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. Unlike platforms that rely on Tn7-medidated transposition for the construction of baculoviruses, HR-bac relies on homologous recombination, which allows to evaluate expression constructs in 2 weeks and is thus perfectly adapted to parallel expression screening. In this chapter, we detail our standard operating procedures for the preparation of the reagents, the construction and evaluation of baculoviruses, and the optimization of protein production for both intracellularly expressed and secreted proteins.
Assuntos
Baculoviridae , Vetores Genéticos , Proteínas Recombinantes , Baculoviridae/genética , Animais , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vetores Genéticos/genética , Células Sf9 , Expressão Gênica , Humanos , Insetos/genética , Spodoptera , Linhagem Celular , Recombinação Homóloga , Análise Custo-BenefícioRESUMO
The inhibitor of κB (IκB) kinase (IKK) is a central regulator of NF-κB signaling. All IKK complexes contain hetero- or homodimers of the catalytic IKKß and/or IKKα subunits. Here, we identify a YDDΦxΦ motif, which is conserved in substrates of canonical (IκBα, IκBß) and alternative (p100) NF-κB pathways, and which mediates docking to catalytic IKK dimers. We demonstrate a quantitative correlation between docking affinity and IKK activity related to IκBα phosphorylation/degradation. Furthermore, we show that phosphorylation of the motif's conserved tyrosine, an event previously reported to promote IκBα accumulation and inhibition of NF-κB gene expression, suppresses the docking interaction. Results from integrated structural analyzes indicate that the motif binds to a groove at the IKK dimer interface. Consistently, suppression of IKK dimerization also abolishes IκBα substrate binding. Finally, we show that an optimized bivalent motif peptide inhibits NF-κB signaling. This work unveils a function for IKKα/ß dimerization in substrate motif recognition.
Assuntos
Motivos de Aminoácidos , Quinase I-kappa B , NF-kappa B , Multimerização Proteica , Quinase I-kappa B/metabolismo , Quinase I-kappa B/química , Quinase I-kappa B/genética , Humanos , NF-kappa B/metabolismo , Fosforilação , Ligação Proteica , Transdução de Sinais , Inibidor de NF-kappaB alfa/metabolismo , Inibidor de NF-kappaB alfa/genética , Simulação de Acoplamento Molecular , Células HEK293 , Especificidade por SubstratoRESUMO
Rapid preparation of proteins for functional and structural analysis is a major challenge both in academia and industry. The number potential targets continuously increases and many are difficult to express proteins which, when produced in bacteria, result in insoluble and/or misfolded recombinant proteins, protein aggregates, or unusable low protein yield. We focus here on the baculovirus expression vector system which is now commonly used for heterologous production of human targets. This chapter describes simple and cost-effective protocols that enable iterative cycles of construct design, expression screening and optimization of protein production. We detail time- and cost-effective methods for generation of baculoviruses by homologous recombination and titer evaluation. Handling of insect cell cultures and preparation of bacmid for cotransfection are also presented.
Assuntos
Baculoviridae , Vetores Genéticos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Técnicas de Cultura de Células , Vetores Genéticos/genética , Humanos , Insetos/genética , Insetos/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
The Baculovirus/insect cell expression system is a powerful technology for reconstitution of eukaryotic macromolecular assemblies. Most multigene expression platforms rely on Tn7-mediated transposition for transferring the expression cassette into the baculoviral genome. This allows a rigorous characterization of recombinant bacmids but involves multiple steps, a limitation when many constructs are to be tested. For parallel expression screening and potential high throughput applications, we have established an open source multigene-expression toolbox exploiting homologous recombination, thus reducing the recombinant baculovirus generation to a single-step procedure and shortening the time from cloning to protein production to 2 weeks. The HR-bac toolbox is composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. They contain single or dual expression cassettes bearing different affinity tags and their design facilitates the mix and match utilization of expression units from Multibac constructs. The overall cost of virus generation with HR-bac toolbox is relatively low as the preparation of linearized baculoviral DNA only requires standard reagents. Various multiprotein assemblies (nuclear hormone receptor heterodimers, the P-TEFb or the ternary CAK kinase complex associated with the XPD TFIIH subunit) are used as model systems to validate the toolbox presented.
RESUMO
The cuticle of C. elegans is impermeable to chemicals, toxins, and pathogens. However, increased permeability is a desirable phenotype because it facilitates chemical uptake. Surface lipids contribute to the permeability barrier. Here, we identify the lipid transfer protein GMAP-1 as a critical element setting the permeability of the C. elegans cuticle. A gmap-1 deletion mutant increases cuticular permeability to sodium azide, levamisole, Hoechst, and DiI. Expressing GMAP-1 in the hypodermis or transiently in the adults is sufficient to rescue this gmap-1 permeability phenotype. GMAP-1 protein is secreted from the hypodermis to the aqueous fluid filling the space between collagen fibers of the cuticle. In vitro, GMAP-1 protein binds phosphatidylserine and phosphatidylcholine while in vivo, GMAP-1 sets the surface lipid composition and organization. Altogether, our results suggest GMAP-1 secreted by hypodermis shuttles lipids to the surface to form the permeability barrier of C. elegans.
RESUMO
The need to generate modified cell lines that express tagged proteins of interest has become increasingly important. Here, we describe a detailed protocol for facile CRISPR/Cas9-mediated gene tagging and isolation of modified cells. In this protocol, we combine two previously published strategies that promote CRISPR/Cas9-mediated gene tagging: using chemically modified single-stranded oligonucleotides as donor templates and a co-selection strategy targeting the ATP1A1 gene at the same time as the gene of interest. Altogether, the protocol proposed here is both easier and saves time compared to other approaches for generating cells that express tagged proteins of interest, which is crucial to purify native complex from human cells.
Assuntos
Biotecnologia/métodos , Sistemas CRISPR-Cas , Edição de Genes/métodos , Marcação de Genes/métodos , Linhagem Celular , DNA Helicases/biossíntese , DNA Helicases/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Expressão Gênica , Humanos , Células K562 , Oligonucleotídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Fator de Transcrição TFIIH/biossíntese , Fator de Transcrição TFIIH/genética , TransfecçãoRESUMO
Macromolecular complexes govern the majority of biological processes and are of great biomedical relevance as factors that perturb interaction networks underlie a number of diseases, and inhibition of protein-protein interactions is a common strategy in drug discovery. Genome editing technologies enable precise modifications in protein coding genes in mammalian cells, offering the possibility to introduce affinity tags or fluorescent reporters for proteomic or imaging applications in the bona fide cellular context. Here we describe a streamlined procedure which uses the CRISPR/Cas9 system and a double-stranded donor plasmid for efficient generation of homozygous endogenously GFP-tagged human cell lines. Establishing cellular models that preserve native genomic regulation of the target protein is instrumental to investigate protein localization and dynamics using fluorescence imaging but also to affinity purify associated protein complexes using anti-GFP antibodies or nanobodies.
Assuntos
Sistemas CRISPR-Cas , DNA/genética , Edição de Genes , Proteínas de Fluorescência Verde/genética , Proteínas Recombinantes de Fusão/genética , Sequência de Bases , Clonagem Molecular , Citometria de Fluxo , Expressão Gênica , Marcação de Genes , Células HEK293 , Humanos , Microscopia de Fluorescência , Modelos Moleculares , Plasmídeos/genética , Conformação Proteica , RNA Guia de Cinetoplastídeos , Proteínas Recombinantes de Fusão/química , Relação Estrutura-AtividadeRESUMO
Toxoplasmosis can be a life-threatening infection, particularly during pregnancy and in immunocompromised patients. The biological diagnosis of toxoplasmosis is challenging and has been revolutionized by molecular detection methods. This article summarizes the data of a multicenter study involving four centers to assess the performances of a commercial PCR assay as compared with four in-house PCR assays using Toxoplasma gondii standards, 20 external quality control specimens, and 133 clinical samples. This clinical cohort includes well-characterized clinical samples corresponding to different clinical situations: confirmed congenital toxoplasmosis (44 samples), toxoplasmosis in immunocompromised patients (25 samples), and chorioretinitis (5 samples). Furthermore, 59 samples from patients without toxoplasmosis were included as negative controls. The analytical sensitivities of the five methods tested were very similar; and the limit of Toxoplasma DNA detection was around 0.01 T. gondii genome per reaction for all the methods. The overall concordance between the commercial PCR and the four in-house PCR assays was 97.7% (130/133). The clinical sensitivity and specificity were >98% and could be increased for the commercial kit when PCR was performed in multiplicate to detect low parasitic loads. In conclusion, the commercial PCR assay shows suitable performances to diagnose the different clinical forms of toxoplasmosis.