RESUMO
We report a tool for the analysis of subcellular proteomics data, called MetaMass, based on the use of standardized lists of subcellular markers. We analyzed data from 11 studies using MetaMass, mapping the subcellular location of 5,970 proteins. Our analysis revealed large variations in the performance of subcellular fractionation protocols as well as systematic biases in protein annotation databases. The Excel and R versions of MetaMass should enhance transparency and reproducibility in subcellular proteomics.
Assuntos
Metanálise como Assunto , Proteínas/metabolismo , Proteômica/métodos , Frações Subcelulares/metabolismo , Algoritmos , Animais , Biomarcadores/metabolismo , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Camundongos , Proteômica/estatística & dados numéricosRESUMO
Lung cancer is often refractory to radiotherapy, but molecular mechanisms of tumor resistance remain poorly defined. Here we show that the receptor tyrosine kinase EphA5 is specifically overexpressed in lung cancer and is involved in regulating cellular responses to genotoxic insult. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ATM (ataxia-telangiectasia mutated) at sites of DNA repair. Finally, we demonstrate that a new monoclonal antibody against human EphA5 sensitized lung cancer cells and human lung cancer xenografts to radiotherapy and significantly prolonged survival, thus suggesting the likelihood of translational applications.
Assuntos
Neoplasias Pulmonares/enzimologia , Receptor EphA5/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Ciclo Celular , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia de Alvo Molecular , Tolerância a Radiação , Ratos , Ratos Nus , Receptor EphA5/imunologiaRESUMO
In this article, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization.
Assuntos
Proteínas de Fluorescência Verde/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Amiloide/química , Bioensaio , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Temperatura Alta , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Eletricidade EstáticaRESUMO
Microorganisms that degrade biomass produce diverse assortments of carbohydrate-active enzymes and binding modules. Despite tremendous advances in the genomic sequencing of these organisms, many genes do not have an ascribed function owing to low sequence identity to genes that have been annotated. Consequently, biochemical and structural characterization of genes with unknown function is required to complement the rapidly growing pool of genomic sequencing data. A protein with previously unknown function (Cthe_2159) was recently isolated in a genome-wide screen using phage display to identify cellulose-binding protein domains from the biomass-degrading bacterium Clostridium thermocellum. Here, the crystal structure of Cthe_2159 is presented and it is shown that it is a unique right-handed parallel ß-helix protein. Despite very low sequence identity to known ß-helix or carbohydrate-active proteins, Cthe_2159 displays structural features that are very similar to those of polysaccharide lyase (PL) families 1, 3, 6 and 9. Cthe_2159 is conserved across bacteria and some archaea and is a member of the domain of unknown function family DUF4353. This suggests that Cthe_2159 is the first representative of a previously unknown family of cellulose and/or acid-sugar binding ß-helix proteins that share structural similarities with PLs. Importantly, these results demonstrate how functional annotation by biochemical and structural analysis remains a critical tool in the characterization of new gene products.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cálcio/química , Cálcio/metabolismo , Celulose/metabolismo , Clonagem Molecular , Clostridium thermocellum/química , Clostridium thermocellum/metabolismo , Cristalografia por Raios X , Gadolínio/química , Modelos Moleculares , Polissacarídeo-Liases/química , Conformação Proteica , Estrutura Terciária de Proteína , Homologia Estrutural de ProteínaRESUMO
Antibodies are used in many areas of biomedical and clinical research, but many of these antibodies have not been adequately characterized, which casts doubt on the results reported in many scientific papers. This problem is compounded by a lack of suitable control experiments in many studies. In this article we review the history of the 'antibody characterization crisis', and we document efforts and initiatives to address the problem, notably for antibodies that target human proteins. We also present recommendations for a range of stakeholders - researchers, universities, journals, antibody vendors and repositories, scientific societies and funders - to increase the reproducibility of studies that rely on antibodies.
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Anticorpos , Pesquisa Biomédica , Reprodutibilidade dos Testes , Humanos , AnimaisRESUMO
BACKGROUND: Single cell genomics has revolutionized microbial sequencing, but complete coverage of genomes in complex microbiomes is imperfect due to enormous variation in organismal abundance and amplification bias. Empirical methods that complement rapidly improving bioinformatic tools will improve characterization of microbiomes and facilitate better genome coverage for low abundance microbes. METHODS: We describe a new approach to sequencing individual species from microbiomes that combines antibody phage display against intact bacteria with fluorescence activated cell sorting (FACS). Single chain (scFv) antibodies are selected using phage display against a bacteria or microbial community, resulting in species-specific antibodies that can be used in FACS for relative quantification of an organism in a community, as well as enrichment or depletion prior to genome sequencing. RESULTS: We selected antibodies against Lactobacillus acidophilus and demonstrate a FACS-based approach for identification and enrichment of the organism from both laboratory-cultured and commercially derived bacterial mixtures. The ability to selectively enrich for L. acidophilus when it is present at a very low abundance (<0.2%) leads to complete (>99.8%) de novo genome coverage whereas the standard single-cell sequencing approach is incomplete (<68%). We show that specific antibodies can be selected against L. acidophilus when the monoculture is used as antigen as well as when a community of 10 closely related species is used demonstrating that in principal antibodies can be generated against individual organisms within microbial communities. CONCLUSIONS: The approach presented here demonstrates that phage-selected antibodies against bacteria enable identification, enrichment of rare species, and depletion of abundant organisms making it tractable to virtually any microbe or microbial community. Combining antibody specificity with FACS provides a new approach for characterizing and manipulating microbial communities prior to genome sequencing.
Assuntos
Anticorpos Antibacterianos/metabolismo , Carga Bacteriana/métodos , Citometria de Fluxo/métodos , Lactobacillus acidophilus/isolamento & purificação , Microbiota , Análise de Sequência de DNA/métodos , Anticorpos de Cadeia Única/metabolismo , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/isolamento & purificação , Técnicas de Visualização da Superfície Celular , Lactobacillus acidophilus/genética , Lactobacillus acidophilus/imunologia , Dados de Sequência Molecular , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/isolamento & purificaçãoRESUMO
The therapeutic use of monoclonal antibodies (mAbs) ranges from cancer treatment to immune-mediated conditions, covering infectious and cardiovascular disorders, among others. The development of improved methods for therapeutic antibody discovery has accelerated the identification of numerous mAbs: a discovery campaign can be deeply mined, resulting in hundreds, even thousands, of potential antibody leads for a given target of interest. High throughput mAb expression and purification methods are required for the rapid validation of those leads. In this work, we describe the implementation of a Protein-A coated membrane plate system, the Purexa™ AHT membrane plate, for robust preparative purification of hundreds of recombinant mAbs, without the need for automation. The high efficiency (>80%) recovery generated sufficient mAb for downstream screening analyses such as ELISA and surface plasmon resonance (SPR). This new system allows the functional validation of hundreds of lead antibodies from discovery campaigns in a timely manner regardless of operational size.
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Anticorpos Monoclonais , Proteína Estafilocócica A , Proteínas Recombinantes , Ressonância de Plasmônio de Superfície , Ensaio de Imunoadsorção EnzimáticaRESUMO
Accurate and efficient affinity measurement techniques are essential for the biophysical characterization of therapeutic monoclonal antibodies, one of the fastest growing drug classes. Surface plasmon resonance (SPR) is widely used for determining antibody affinity, but does not perform well with extremely high affinity (low picomolar to femtomolar range) molecules. In this study, we compare the SPR-based Carterra LSA and the kinetic exclusion assay (KinExA) for measuring the affinities of 48 antibodies generated against the SARS-CoV-2 receptor-binding domain. These data reveal that high-affinity antibodies can be generated straight from selections using high-quality in vitro library platforms with 54% correspondence between affinities measured using LSA and KinExA. Generally, where there was a 2-fold or greater difference between LSA and KinExA, KinExA reported that affinities were tighter. We highlight the differences between LSA and KinExA, identifying the benefits and pitfalls of each in terms of dynamic range and throughput. Furthermore, we demonstrate for the first time that single-point screening with KinExA can significantly improve throughput while maintaining a strong correlation with full binding curve equilibrium measurements, enabling the accurate rank-ordering of clones with exceptionally tight binding properties.
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Anticorpos Monoclonais , Ressonância de Plasmônio de Superfície , Ressonância de Plasmônio de Superfície/métodos , Anticorpos Monoclonais/química , Afinidade de AnticorposRESUMO
Antibodies are important reagents for research, diagnostics, and therapeutics. Many examples of chimeric proteins combining the specific target recognition of antibodies with complementing functionalities such as fluorescence, toxicity or enzymatic activity have been described. However, antibodies selected solely on the basis of their binding specificities are not necessarily ideal candidates for the construction of chimeras. Here, we describe a high throughput method based on yeast display to directly select antibodies most suitable for conversion to fluorescent chimera. A library of scFv binders was converted to a fluorescent chimeric form, by cloning thermal green protein into the linker between VH and VL, and directly selecting for both binding and fluorescent functionality. This allowed us to directly identify antibodies functional in the single chain TGP format, that manifest higher protein expression, easier protein purification, and one-step binding assays.
Assuntos
Saccharomyces cerevisiae , Anticorpos de Cadeia Única , Saccharomyces cerevisiae/metabolismo , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/genética , Anticorpos Monoclonais , ImunofluorescênciaRESUMO
Therapeutic antibody discovery often relies on in-vitro display methods to identify lead candidates. Assessing selected output diversity traditionally involves random colony picking and Sanger sequencing, which has limitations. Next-generation sequencing (NGS) offers a cost-effective solution with increased read depth, allowing a comprehensive understanding of diversity. Our study establishes NGS guidelines for antibody drug discovery, demonstrating its advantages in expanding the number of unique HCDR3 clusters, broadening the number of high affinity antibodies, expanding the total number of antibodies recognizing different epitopes, and improving lead prioritization. Surprisingly, our investigation into the correlation between NGS-derived frequencies of CDRs and affinity revealed a lack of association, although this limitation could be moderately mitigated by leveraging NGS clustering, enrichment and/or relative abundance across different regions to enhance lead prioritization. This study highlights NGS benefits, offering insights, recommendations, and the most effective approach to leverage NGS in therapeutic antibody discovery.
Assuntos
Anticorpos , Sequenciamento de Nucleotídeos em Larga Escala , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Anticorpos/genética , EpitoposRESUMO
Determining transcription factor (TF) recognition motifs or operator sites is central to understanding gene regulation, yet few operators have been characterized. In this study, we used a protein-binding microarray (PBM) to discover the DNA recognition sites and putative regulons for three TetR and one MarR family TFs derived from Burkholderia xenovorans, which are common to the genus Burkholderia. We also describe the development and application of a more streamlined version of the PBM technology that significantly reduced the experimental time. Despite the genus containing many pathogenically important species, only a handful of TF operator sites have been experimentally characterized for Burkholderia to date. Our study provides a significant addition to this knowledge base and illustrates some general challenges of discovering operators on a large scale for prokaryotes.
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Proteínas de Bactérias/genética , Burkholderia/genética , Regiões Operadoras Genéticas , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Burkholderia/química , Burkholderia/classificação , Burkholderia/metabolismo , Dados de Sequência Molecular , Família Multigênica , Filogenia , Ligação Proteica , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one on-line warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site.
Assuntos
Bases de Dados de Proteínas/normas , Proteoma/análise , Sistemas de Gerenciamento de Base de Dados/normas , Humanos , Cooperação Internacional , Proteômica/métodos , Terminologia como AssuntoRESUMO
Filamentous phage display has been extensively used to select proteins with binding properties of specific interest. Although many different display platforms using filamentous phage have been described, no comprehensive comparison of their abilities to display similar proteins has been conducted. This is particularly important for the display of cytoplasmic proteins, which are often poorly displayed with standard filamentous phage vectors. In this article, we have analyzed the ability of filamentous phage to display a stable form of green fluorescent protein and modified variants in nine different display vectors, a number of which have been previously proposed as being suitable for cytoplasmic protein display. Correct folding and display were assessed by phagemid particle fluorescence, and with anti-GFP antibodies. The poor correlation between phagemid particle fluorescence and recognition of GFP by antibodies, indicates that proteins may fold correctly without being accessible for display. The best vector used a twin arginine transporter leader to transport the displayed protein to the periplasm, and a coil-coil arrangement to link the displayed protein to g3p. This vector was able to display less robust forms of GFP, including ones with inserted epitopes, as well as fluorescent proteins of the Azami green series. It was also functional in mock selection experiments.
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Corantes Fluorescentes/análise , Vetores Genéticos , Proteínas de Fluorescência Verde/análise , Inovirus/genética , Citoplasma/química , Proteínas de Fluorescência Verde/genética , Plasmídeos/químicaRESUMO
We have developed a high-throughput protein expression and interaction analysis platform that combines cDNA phage display library selection and massive gene sequencing using the 454 platform. A phage display library of open reading frame (ORF) fragments was created from mRNA derived from different tissues. This was used to study the interaction network of the enzyme transglutaminase 2 (TG2), a multifunctional enzyme involved in the regulation of cell growth, differentiation and apoptosis, associated with many different pathologies. After two rounds of panning with TG2 we assayed the frequency of ORFs within the selected phage population using 454 sequencing. Ranking and analysis of more than 120,000 sequences allowed us to identify several potential interactors, which were subsequently confirmed in functional assays. Within the identified clones, three had been previously described as interacting proteins (fibronectin, SMOC1 and GSTO2), while all the others were new. When compared with standard systems, such as microtiter enzyme-linked immunosorbant assay, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Análise de Sequência de DNA/métodos , DNA Complementar/química , Proteínas de Ligação ao GTP/metabolismo , Humanos , Fases de Leitura Aberta , Biblioteca de Peptídeos , Proteína 2 Glutamina gama-Glutamiltransferase , Domínios e Motivos de Interação entre Proteínas , Transglutaminases/metabolismoRESUMO
The intense international focus on the COVID-19 pandemic has provided a unique opportunity to use a wide array of novel tools to carry out scientific studies on the SARS-CoV-2 virus. The value of these comparative studies extends far beyond their consequences for SARS-CoV-2, providing broad implications for health-related science. Here we specifically discuss the impacts of these comparisons on advances in vaccines, the analysis of host humoral immunity, and antibody discovery. As an extension, we also discuss potential synergies between these areas.Abbreviations: CoVIC: The Coronavirus Immunotherapeutic Consortium; EUA: Emergency Use Authorization.
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COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , COVID-19/epidemiologia , Humanos , Imunidade Humoral , Pandemias/prevenção & controleRESUMO
As a result of the SARS-CoV-2 pandemic numerous scientific groups have generated antibodies against a single target: the CoV-2 spike antigen. This has provided an unprecedented opportunity to compare the efficacy of different methods and the specificities and qualities of the antibodies generated by those methods. Generally, the most potent neutralizing antibodies have been generated from convalescent patients and immunized animals, with non-immune phage libraries usually yielding significantly less potent antibodies. Here, we show that it is possible to generate ultra-potent (IC50 < 2 ng/ml) human neutralizing antibodies directly from a unique semisynthetic naïve antibody library format with affinities, developability properties and neutralization activities comparable to the best from hyperimmune sources. This demonstrates that appropriately designed and constructed naïve antibody libraries can effectively compete with immunization to directly provide therapeutic antibodies against a viral pathogen, without the need for immune sources or downstream optimization.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos/imunologia , COVID-19/epidemiologia , COVID-19/virologia , Chlorocebus aethiops , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Testes de Neutralização/métodos , Pandemias , Biblioteca de Peptídeos , Ligação Proteica , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células VeroRESUMO
ABBREVIATIONS: CDR: complementarity determining region; FACS: fluorescence-activated cell sorting; ka: association rate; kd: dissociation rate; KD: dissociation constant; scFv: single-chain variable fragment; SPR: surface plasmon resonance.
Assuntos
Anticorpos de Cadeia Única , Afinidade de Anticorpos , Regiões Determinantes de Complementaridade , Ressonância de Plasmônio de SuperfícieRESUMO
BACKGROUND: In order to carry out experimental gene annotation, DNA encoding open reading frames (ORFs) derived from real genes (termed "genic") in the correct frame is required. When genes are correctly assigned, isolation of genic DNA for functional annotation can be carried out by PCR. However, not all genes are correctly assigned, and even when correctly assigned, gene products are often incorrectly folded when expressed in heterologous hosts. This is a problem that can sometimes be overcome by the expression of protein fragments encoding domains, rather than full-length proteins. One possible method to isolate DNA encoding such domains would to "filter" complex DNA (cDNA libraries, genomic and metagenomic DNA) for gene fragments that confer a selectable phenotype relying on correct folding, with all such domains present in a complex DNA sample, termed the "domainome". RESULTS: In this paper we discuss the preparation of diverse genic ORF libraries from randomly fragmented genomic DNA using ß-lactamase to filter out the open reading frames. By cloning DNA fragments between leader sequences and the mature ß-lactamase gene, colonies can be selected for resistance to ampicillin, conferred by correct folding of the lactamase gene. Our experiments demonstrate that the majority of surviving colonies contain genic open reading frames, suggesting that ß-lactamase is acting as a selectable folding reporter. Furthermore, different leaders (Sec, TAT and SRP), normally translocating different protein classes, filter different genic fragment subsets, indicating that their use increases the fraction of the "domainone" that is accessible. CONCLUSIONS: The availability of ORF libraries, obtained with the filtering method described here, combined with screening methods such as phage display and protein-protein interaction studies, or with protein structure determination projects, can lead to the identification and structural determination of functional genic ORFs. ORF libraries represent, moreover, a useful tool to proceed towards high-throughput functional annotation of newly sequenced genomes.
Assuntos
Clostridium thermocellum/genética , Genômica/métodos , Fases de Leitura Aberta , DNA Bacteriano , Biblioteca Gênica , Anotação de Sequência Molecular , Análise de Sequência de DNA , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Recent recommendations from the European Union Reference Laboratory regarding the generation of antibodies using animals have stimulated significant debate. Here, four of the scientists who served on the Scientific Advisory Committee provide clarification of their views regarding the use of animals and in vitro platforms in antibody generation.Abbreviations: EURL ECVAM, European Union Reference Laboratory for alternatives to animal testing. ESAC, EURL ECVAM Scientific Advisory Committee.