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Philippe Lamesch - One of the best experts on this subject based on the ideXlab platform.

  • hORFeome v3 1 a resource of human open reading frames representing over 10 000 human genes
    Genomics, 2007
    Co-Authors: Philippe Lamesch, Stuart Milstein, Zhenjun Hu, Kavitha Venkatesan, Graeme Bethel, Paul Martin, Gabor Szabo, Ning Li, Jane Rogers
    Abstract:

    Complete sets of cloned protein-encoding open reading frames (ORFs), or ORFeomes, are essential tools for large-scale proteomics and systems biology studies. Here we describe human ORFeome version 3.1 (hORFeome v3.1), currently the largest publicly available resource of full-length human ORFs (available at www.openbiosystems.com). Generated by Gateway recombinational cloning, this collection contains 12,212 ORFs, representing 10,214 human genes, and corresponds to a 51% expansion of the original hORFeome v1.1. An online human ORFeome database, hORFDB, was built and serves as the central repository for all cloned human ORFs (http://horfdb.dfci.harvard.edu). This expansion of the original ORFeome resource greatly increases the potential experimental search space for large-scale proteomics studies, which will lead to the generation of more comprehensive datasets.

  • Closing in on the C. elegans ORFeome by cloning TWINSCAN predictions
    Genome research, 2005
    Co-Authors: Chaochun Wei, Philippe Lamesch, Marc Vidal, Manimozhiyan Arumugam, Jennifer Rosenberg, Michael R. Brent
    Abstract:

    The genome of Caenorhabditis elegans was the first animal genome to be sequenced. Although considerable effort has been devoted to annotating it, the standard WormBase annotation contains thousands of predicted genes for which there is no cDNA or EST evidence. We hypothesized that a more complete experimental annotation could be obtained by creating a more accurate gene-prediction program and then amplifying and sequencing predicted genes. Our approach was to adapt the TWINSCAN gene prediction system to C. elegans and C. briggsae and to improve its splice site and intron-length models. The resulting system has 60% sensitivity and 58% specificity in exact prediction of open reading frames (ORFs), and hence, proteins-the best results we are aware of any multicellular organism. We then attempted to amplify, clone, and sequence 265 TWINSCAN-predicted ORFs that did not overlap WormBase gene annotations. The success rate was 55%, adding 146 genes that were completely absent from WormBase to the ORF clone collection (ORFeome). The same procedure had a 7% success rate on 90 Worm Base "predicted" genes that do not overlap TWINSCAN predictions. These results indicate that the accuracy of WormBase could be significantly increased by replacing its partially curated predicted genes with TWINSCAN predictions. The technology described in this study will continue to drive the C. elegans ORFeome toward completion and contribute to the annotation of the three Caenorhabditis species currently being sequenced. The results also suggest that this technology can significantly improve our knowledge of the "parts list" for even the best-studied model organisms.

  • human ORFeome version 1 1 a platform for reverse proteomics
    Genome Research, 2004
    Co-Authors: Jaen Francois Rual, Philippe Lamesch, Tomoko Hirozanekishikawa, Jennifer Rosenberg, Tong Hao, Nicolas Bertin, Amelie Dricot, Pierreolivier Vidalain, Tracey R Clingingsmith, James L Hartley
    Abstract:

    The advent of systems biology necessitates the cloning of nearly entire sets of protein-encoding open reading frames (ORFs), or ORFeomes, to allow functional studies of the corresponding proteomes. Here, we describe the generation of a first version of the human ORFeome using a newly improved Gateway recombinational cloning approach. Using the Mammalian Gene Collection (MGC) resource as a starting point, we report the successful cloning of 8076 human ORFs, representing at least 7263 human genes, as mini-pools of PCR-amplified products. These were assembled into the human ORFeome version 1.1 (hORFeome v1.1) collection. After assessing the overall quality of this version, we describe the use of hORFeome v1.1 for heterologous protein expression in two different expression systems at proteome scale. The hORFeome v1.1 represents a central resource for the cloning of large sets of human ORFs in various settings for functional proteomics of many types, and will serve as the foundation for subsequent improved versions of the human ORFeome.

  • generation of the brucella melitensis ORFeome version 1 1
    Genome Research, 2004
    Co-Authors: Amelie Dricot, Philippe Lamesch, Tong Hao, Nicolas Bertin, Jean Francois Rual, Denis Dupuy, Christophe Lambert, Regis Hallez, Jeanmarc Delroisse, Jean Vandenhaute
    Abstract:

    The bacteria of the Brucella genus are responsible for a worldwide zoonosis called brucellosis. They belong to the α-proteobacteria group, as many other bacteria that live in close association with a eukaryotic host. Importantly, the Brucellae are mainly intracellular pathogens, and the molecular mechanisms of their virulence are still poorly understood. Using the complete genome sequence of Brucella melitensis, we generated a database of protein-coding open reading frames (ORFs) and constructed an ORFeome library of 3091 Gateway Entry clones, each containing a defined ORF. This first version of the Brucella ORFeome (v1.1) provides the coding sequences in a user-friendly format amenable to high-throughput functional genomic and proteomic experiments, as the ORFs are conveniently transferable from the Entry clones to various Expression vectors by recombinational cloning. The cloning of the Brucella ORFeome v1.1 should help to provide a better understanding of the molecular mechanisms of virulence, including the identification of bacterial protein-protein interactions, but also interactions between bacterial effectors and their host's targets.

  • c elegans ORFeome version 3 1 increasing the coverage of ORFeome resources with improved gene predictions
    Genome Research, 2004
    Co-Authors: Philippe Lamesch, Stuart Milstein, Jean Vandenhaute, David E Hill, Jennifer Rosenberg, Tong Hao, Reynaldo Sequerra, Stephanie Bosak, Lynn Doucettestamm, Marc Vidal
    Abstract:

    The Caenorhabditis elegans genome sequence, released in December 1998, was nearly complete and highly accurate, with an error rate estimated at 1/30,000 (The C. elegans Sequencing Consortium 1998). The finished sequence was eventually released in November 2002, comprising 100,258,171 bp in six contiguous segments corresponding to the six C. elegans chromosomes (J. Sulston, pers com; http://elegans.swmed.edu/Announcements/genome_complete.html). Although the technology required for rapid and accurate whole-genome sequencing is mature, the gene prediction tools currently available to identify protein-encoding open reading frames (ORFs) and to define their exon/intron structures still need improvements. For exon prediction in mammalian genomes, these tools have an overall sensitivity and specificity of only 60% (Burset and Guigo 1996), and ∼40% for the 5′ and 3′ gene boundaries specifically (Korf et al. 2001). Predicted genes can be truncated, extended, split, or merged (see Reboul et al. 2001), relative to their actual “observed” exon/intron structure. Using GeneFinder, a gene prediction tool developed for C. elegans (http://ftp.genome.washington.edu/cgi-bin/genefinder_req.pl), a total of 19,477 ORFs were annotated in Wormbase release WS9 (August 1999; http://www.Wormbase.org; Stein et al. 2001). Approximately 50% of these ORFs were predicted ab initio, without experimental support. The C. elegans ORFeome project was launched to test the accuracy of these gene predictions, while simultaneously creating a resource of cloned full-length predicted ORFs to be used in various functional genomics and reverse proteomics studies (Reboul et al. 2001, 2003). ORFs were PCR-amplified between their 5′- and 3′-ends, and cloned using the Gateway recombinational cloning system (Hartley et al. 2000; Walhout et al. 2000a,b). PCR amplification was performed on a highly representative cDNA library using gene-specific primer pairs for each of the 19,477 ORFs based on WS9 predictions. Gateway tails attached to all primers allowed the cloning of the ORFs into the pDONR201 vector, resulting in a total of 11,984 (61.5% of the ORFs) Entry clones in the first version of the ORFeome (version v1.1; Supplemental Table 1). The C. elegans ORFeome version 1.1a (v1.1a) represents a consolidated set of 10,623 ORFs cloned in-frame, 11.4% (1361 out of 11,984) of all cloned ORFs in version 1 were cloned outof-frame because of mispredicted gene boundaries (v1.1b). This first version of the worm ORFeome contributed significantly to the reannotation of C. elegans gene structure. The alignment of OSTs (ORF Sequence Tags) to the corresponding predicted gene sequences allowed the improvement of C. elegans annotations by correcting the internal gene structure of 20% of v1.1a cloned ORFs. In addition, OSTs provided experimental verification for 45% of the set of “untouched” ORFs, that is, not detected yet by any mRNA or EST. For each gene, ORFeome v1.1a contains cloned pools that result from mixing ∼50 to ∼1000 Escherichia coli transformants for each Entry clone. Thus, such Entry pools might contain multiple splice variants and alleles corresponding to PCR misincorporations. We are in the process of generating a new resource, ORFeome v2 (Reboul et al. 2003), in which we isolate individual wild-type clones for all detected splice variants of ORFs cloned in v1.1a. We will shortly initiate similar attempts for the ORFs cloned in the ORFeome version 3 described below. The difficulties inherent in identifying ORFs within metazoan genomes and predicting their correct structure are not specific to C. elegans. Genome annotation initiatives in the model organisms Arabidopsis thaliana (Yamada et al. 2003) and Drosophila melanogaster (Hild et al. 2003) have also shown limited accuracy. The accuracy of current gene prediction algorithms is also a major issue for the human genome. High numbers of splice variants and lower signal-to-noise ratios caused by longer introns and intergenic regions render human genome annotations even more difficult than for the model systems experimentally validated so far. Hence, both in model organisms and in human, functional genomic and reverse proteomics studies, which require the use of large sets of full-length ORFs, are hampered by inaccuracies in gene prediction, limiting the usefulness of sequenced genomes. Since the release of Wormbase WS9 in 1999, continuous efforts to reannotate the C. elegans genome have occurred. Reannotations are mainly based on new experimental data, such as mRNAs and ESTs (the EMBL nucleotide sequence database [http://www.ebi.ac.uk/embl/] and the Y. Kohara DNA databank [DDBJ, http://www.ddbj.nig.ac.jp/]), as well as splice-leader sequences (Blumenthal et al. 2002). Furthermore, more refined ab initio approaches have allowed the reprediction of genes for which no confirmatory experimental data are yet available. To experimentally validate these new predictions, improve gene annotation, and generate a more complete C. elegans ORFeome resource, we attempted to clone the 4232 ORFs originally missed in v1.1a and that have been either repredicted or newly predicted between the release of WS9 and that of WS100 (May 2003).

Marc Vidal - One of the best experts on this subject based on the ideXlab platform.

  • Closing in on the C. elegans ORFeome by cloning TWINSCAN predictions
    Genome research, 2005
    Co-Authors: Chaochun Wei, Philippe Lamesch, Marc Vidal, Manimozhiyan Arumugam, Jennifer Rosenberg, Michael R. Brent
    Abstract:

    The genome of Caenorhabditis elegans was the first animal genome to be sequenced. Although considerable effort has been devoted to annotating it, the standard WormBase annotation contains thousands of predicted genes for which there is no cDNA or EST evidence. We hypothesized that a more complete experimental annotation could be obtained by creating a more accurate gene-prediction program and then amplifying and sequencing predicted genes. Our approach was to adapt the TWINSCAN gene prediction system to C. elegans and C. briggsae and to improve its splice site and intron-length models. The resulting system has 60% sensitivity and 58% specificity in exact prediction of open reading frames (ORFs), and hence, proteins-the best results we are aware of any multicellular organism. We then attempted to amplify, clone, and sequence 265 TWINSCAN-predicted ORFs that did not overlap WormBase gene annotations. The success rate was 55%, adding 146 genes that were completely absent from WormBase to the ORF clone collection (ORFeome). The same procedure had a 7% success rate on 90 Worm Base "predicted" genes that do not overlap TWINSCAN predictions. These results indicate that the accuracy of WormBase could be significantly increased by replacing its partially curated predicted genes with TWINSCAN predictions. The technology described in this study will continue to drive the C. elegans ORFeome toward completion and contribute to the annotation of the three Caenorhabditis species currently being sequenced. The results also suggest that this technology can significantly improve our knowledge of the "parts list" for even the best-studied model organisms.

  • toward improving caenorhabditis elegans phenome mapping with an ORFeome based rnai library
    Genome Research, 2004
    Co-Authors: Jaen Francois Rual, Julian Ceron, John Koreth, Anne Sophie Nicot, Tomoko Hirozanekishikawa, Jean Vandenhaute, Stuart H Orkin, David E Hill, Sander Van Den Heuvel, Marc Vidal
    Abstract:

    The recently completed Caenorhabditis elegans genome sequence allows application of high-throughput (HT) approaches for phenotypic analyses using RNA interference (RNAi). As large phenotypic data sets become available, “phenoclustering” strategies can be used to begin understanding the complex molecular networks involved in development and other biological processes. The current HT-RNAi resources represent a great asset for phenotypic profiling but are limited by lack of flexibility. For instance, existing resources do not take advantage of the latest improvements in RNAi technology, such as inducible hairpin RNAi. Here we show that a C. elegans ORFeome resource, generated with the Gateway cloning system, can be used as a starting point to generate alternative HT-RNAi resources with enhanced flexibility. The versatility inherent to the Gateway system suggests that additional HT-RNAi libraries can now be readily generated to perform gene knockdowns under various conditions, increasing the possibilities for phenome mapping in C. elegans.

  • ORFeome cloning and systems biology standardized mass production of the parts from the parts list
    Genome Research, 2004
    Co-Authors: Michael A Brasch, James L Hartley, Marc Vidal
    Abstract:

    Together with metabolites, proteins and RNAs form complex biological systems through highly intricate networks of physical and functional interactions. Large-scale studies aimed at a molecular understanding of the structure, function, and dynamics of proteins and RNAs in the context of cellular networks require novel approaches and technologies. This Special Issue of Genome Research features strategies for the high-throughput construction and manipulation of complete sets of protein-encoding open reading frames (ORFeome), gene promoters (promoterome), and noncoding RNAs, as predicted from genome and transcriptome sequences. Here we discuss the use of a recombinational cloning system that allows efficiency, adaptability, and compatibility in the generation of ORFeome, promoterome, and other resources.

  • c elegans ORFeome version 3 1 increasing the coverage of ORFeome resources with improved gene predictions
    Genome Research, 2004
    Co-Authors: Philippe Lamesch, Stuart Milstein, Jean Vandenhaute, David E Hill, Jennifer Rosenberg, Tong Hao, Reynaldo Sequerra, Stephanie Bosak, Lynn Doucettestamm, Marc Vidal
    Abstract:

    The Caenorhabditis elegans genome sequence, released in December 1998, was nearly complete and highly accurate, with an error rate estimated at 1/30,000 (The C. elegans Sequencing Consortium 1998). The finished sequence was eventually released in November 2002, comprising 100,258,171 bp in six contiguous segments corresponding to the six C. elegans chromosomes (J. Sulston, pers com; http://elegans.swmed.edu/Announcements/genome_complete.html). Although the technology required for rapid and accurate whole-genome sequencing is mature, the gene prediction tools currently available to identify protein-encoding open reading frames (ORFs) and to define their exon/intron structures still need improvements. For exon prediction in mammalian genomes, these tools have an overall sensitivity and specificity of only 60% (Burset and Guigo 1996), and ∼40% for the 5′ and 3′ gene boundaries specifically (Korf et al. 2001). Predicted genes can be truncated, extended, split, or merged (see Reboul et al. 2001), relative to their actual “observed” exon/intron structure. Using GeneFinder, a gene prediction tool developed for C. elegans (http://ftp.genome.washington.edu/cgi-bin/genefinder_req.pl), a total of 19,477 ORFs were annotated in Wormbase release WS9 (August 1999; http://www.Wormbase.org; Stein et al. 2001). Approximately 50% of these ORFs were predicted ab initio, without experimental support. The C. elegans ORFeome project was launched to test the accuracy of these gene predictions, while simultaneously creating a resource of cloned full-length predicted ORFs to be used in various functional genomics and reverse proteomics studies (Reboul et al. 2001, 2003). ORFs were PCR-amplified between their 5′- and 3′-ends, and cloned using the Gateway recombinational cloning system (Hartley et al. 2000; Walhout et al. 2000a,b). PCR amplification was performed on a highly representative cDNA library using gene-specific primer pairs for each of the 19,477 ORFs based on WS9 predictions. Gateway tails attached to all primers allowed the cloning of the ORFs into the pDONR201 vector, resulting in a total of 11,984 (61.5% of the ORFs) Entry clones in the first version of the ORFeome (version v1.1; Supplemental Table 1). The C. elegans ORFeome version 1.1a (v1.1a) represents a consolidated set of 10,623 ORFs cloned in-frame, 11.4% (1361 out of 11,984) of all cloned ORFs in version 1 were cloned outof-frame because of mispredicted gene boundaries (v1.1b). This first version of the worm ORFeome contributed significantly to the reannotation of C. elegans gene structure. The alignment of OSTs (ORF Sequence Tags) to the corresponding predicted gene sequences allowed the improvement of C. elegans annotations by correcting the internal gene structure of 20% of v1.1a cloned ORFs. In addition, OSTs provided experimental verification for 45% of the set of “untouched” ORFs, that is, not detected yet by any mRNA or EST. For each gene, ORFeome v1.1a contains cloned pools that result from mixing ∼50 to ∼1000 Escherichia coli transformants for each Entry clone. Thus, such Entry pools might contain multiple splice variants and alleles corresponding to PCR misincorporations. We are in the process of generating a new resource, ORFeome v2 (Reboul et al. 2003), in which we isolate individual wild-type clones for all detected splice variants of ORFs cloned in v1.1a. We will shortly initiate similar attempts for the ORFs cloned in the ORFeome version 3 described below. The difficulties inherent in identifying ORFs within metazoan genomes and predicting their correct structure are not specific to C. elegans. Genome annotation initiatives in the model organisms Arabidopsis thaliana (Yamada et al. 2003) and Drosophila melanogaster (Hild et al. 2003) have also shown limited accuracy. The accuracy of current gene prediction algorithms is also a major issue for the human genome. High numbers of splice variants and lower signal-to-noise ratios caused by longer introns and intergenic regions render human genome annotations even more difficult than for the model systems experimentally validated so far. Hence, both in model organisms and in human, functional genomic and reverse proteomics studies, which require the use of large sets of full-length ORFs, are hampered by inaccuracies in gene prediction, limiting the usefulness of sequenced genomes. Since the release of Wormbase WS9 in 1999, continuous efforts to reannotate the C. elegans genome have occurred. Reannotations are mainly based on new experimental data, such as mRNAs and ESTs (the EMBL nucleotide sequence database [http://www.ebi.ac.uk/embl/] and the Y. Kohara DNA databank [DDBJ, http://www.ddbj.nig.ac.jp/]), as well as splice-leader sequences (Blumenthal et al. 2002). Furthermore, more refined ab initio approaches have allowed the reprediction of genes for which no confirmatory experimental data are yet available. To experimentally validate these new predictions, improve gene annotation, and generate a more complete C. elegans ORFeome resource, we attempted to clone the 4232 ORFs originally missed in v1.1a and that have been either repredicted or newly predicted between the release of WS9 and that of WS100 (May 2003).

  • ORFeome projects gateway between genomics and omics
    Current Opinion in Chemical Biology, 2004
    Co-Authors: Jean Francois Rual, David E Hill, Marc Vidal
    Abstract:

    Abstract The availability of entire genome sequences is expected to revolutionize the way in which biology and medicine are conducted for years to come. However, achieving this promise still requires significant effort in the areas of gene annotation, cloning and expression of thousands of known and heretofore unknown protein-encoding genes. Traditional technologies of manipulating genes are too cumbersome and inefficient when one is dealing with more than a few genes at a time. Entire libraries composed of all protein-encoding open reading frames (ORFs) cloned in highly flexible vectors will be needed to take full advantage of the information found in any genome sequence. The creation of such ORFeome resources using novel technologies for cloning and expressing entire proteomes constitutes an effective gateway from whole genome sequencing efforts to downstream ‘omics’ applications.

Akihisa Matsuyama - One of the best experts on this subject based on the ideXlab platform.

  • microarray based target identification using drug hypersensitive fission yeast expressing ORFeome
    Molecular BioSystems, 2011
    Co-Authors: Yuko Arita, Akihisa Matsuyama, Yoko Yashiroda, Shinichi Nishimura, Takeo Usui, Charles Boone, Minoru Yoshida
    Abstract:

    Identification of the cellular target of small molecules is a major challenge to developing biological tools and drug leads. Here we report a novel microarray-based system for identification of the target or the target pathway of small molecules using a set of drug-hypersensitive fission yeast strains that collectively overexpress each gene in the open reading frame-ome. The major advantage of this method is that it provides genome-wide interrogation but requires a relatively small amount of the test compound. Using this system, we identified 28 genes linked to etoposide sensitivity, which included genes for the drug target topoisomerase II and other plausible factors that regulate etoposide tolerance. Thus, our approach can accelerate the process of target identification of small molecules, which has the potential to reveal highly conserved genes of clinical relevance.

  • systematic cloning of an ORFeome using the gateway system
    Methods of Molecular Biology, 2009
    Co-Authors: Akihisa Matsuyama, Minoru Yoshida
    Abstract:

    With the completion of the genome projects, there are increasing demands on the experimental systems that enable to exploit the entire set of protein-coding open reading frames (ORFs), viz. ORFeome, en masse. Systematic proteomic studies based on cloned ORFeomes are called "reverse proteomics," and have been launched in many organisms in recent years. Cloning of an ORFeome is such an attractive way for comprehensive understanding of biological phenomena, but is a challenging and daunting task. However, recent advances in techniques for DNA cloning using site-specific recombination and for high-throughput experimental techniques have made it feasible to clone an ORFeome with the minimum of exertion. The Gateway system is one of such the approaches, employing the recombination reaction of the bacteriophage lambda. Combining traditional DNA manipulation methods with modern technique of the recombination-based cloning system, it is possible to clone an ORFeome of an organism on an individual level.

  • new insights into chemical biology from ORFeome libraries
    Current Opinion in Chemical Biology, 2008
    Co-Authors: Yoko Yashiroda, Akihisa Matsuyama, Minoru Yoshida
    Abstract:

    As the genomes of many organisms have been sequenced, a variety of global analyses, called ‘omics,’ have been initiated. Cloning of the set of all open reading frames encoded by the genome (ORFeome) of an organism is a major challenge, which serves as an indispensable provision before one launches into the ocean of the postgenomic world. A suitable strategy for high-throughput cloning and expression of thousands of genes is crucial to success. Recently developed systems employing site-specific or homologous recombination have made it feasible to manipulate thousands of ORFs en masse. Using these technologies, several recent studies have successfully fished biologically active small molecules and target proteins out of this bountiful ocean.

  • ORFeome cloning and global analysis of protein localization in the fission yeast schizosaccharomyces pombe
    Nature Biotechnology, 2006
    Co-Authors: Akihisa Matsuyama, Ritsuko Arai, Yoko Yashiroda, Atsuko Shirai, Ayako Kamata, Shigeko Sekido, Yumiko Kobayashi, Atsushi Hashimoto, Makiko Hamamoto, Yasushi Hiraoka
    Abstract:

    ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe

  • Corrigendum: ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe
    Nature Biotechnology, 2006
    Co-Authors: Akihisa Matsuyama, Ritsuko Arai, Yoko Yashiroda, Atsuko Shirai, Ayako Kamata, Shigeko Sekido, Yumiko Kobayashi, Atsushi Hashimoto, Makiko Hamamoto, Yasushi Hiraoka
    Abstract:

    Corrigendum: ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe

Michael Hust - One of the best experts on this subject based on the ideXlab platform.

  • discovery of leptospira spp seroreactive peptides using ORFeome phage display
    PLOS Neglected Tropical Diseases, 2019
    Co-Authors: Siti Roszilawati Ramli, Jonas Zantow, Natalia Novoselova, Gustavo Marçal Schmidt Garcia Moreira, Frank Pessler, Marga G A Goris, Van Nguyen, Michael Hust
    Abstract:

    Leptospirosis is an infectious disease that is transmitted from animals to humans. It is associated with a broad range of clinical presentations, and diagnostic tests with high diagnostic accuracy are required in order to enable accurate diagnosis. Leptospirosis is diagnosed by detecting DNA of the pathogen or antibodies against it in patients’ blood; the latter are preferred in resource limited regions, and diagnostics based on peptides (small fragments of proteins) are advantageous because they are inexpensive to produce and more stable in hot climates than full-length proteins. We used a technique called open reading frame phage display to identify peptides from Leptospira spp. that could be used to detect antibodies against them in human blood. In this method, the pathogen’s genome is fragmented, the corresponding peptides displayed on the surfaces of phages (viruses that infect bacteria), and the peptides that bind most strongly to the patients’ antibodies are then selected by screening. Using this method, we identified 2 leptospiral peptides that accurately identified antibodies against Leptospira spp. in sera from patients with leptospirosis. These results are encouraging because they demonstrate that ORFeome phage display may be a powerful tool to develop better diagnostics for leptospirosis for use in less developed areas.

  • Discovery of Leptospira spp. seroreactive peptides using ORFeome phage display
    PLoS Neglected Tropical Diseases, 2019
    Co-Authors: Siti Roszilawati Ramli, Jonas Zantow, Van Kinh Nguyen, Natalia Novoselova, Gustavo Marçal Schmidt Garcia Moreira, Frank Pessler, Marga G A Goris, Michael Hust
    Abstract:

    Background Leptospirosis is the most common zoonotic disease worldwide. The diagnostic performance of a serological test for human leptospirosis is mainly influenced by the antigen used in the test assay. An ideal serological test should cover all serovars of pathogenic leptospires with high sensitivity and specificity and use reagents that are relatively inexpensive to produce and can be used in tropical climates. Peptide-based tests fulfil at least the latter two requirements, and ORFeome phage display has been successfully used to identify immunogenic peptides from other pathogens. Methodology/Principal findings Two ORFeome phage display libraries of the entire Leptospira spp. genomes from five local strains isolated in Malaysia and seven WHO reference strains were constructed. Subsequently, 18 unique Leptospira peptides were identified in a screen using a pool of sera from patients with acute leptospirosis. Five of these were validated by titration ELISA using different pools of patient or control sera. The diagnostic performance of these five peptides was then assessed against 16 individual sera from patients with acute leptospirosis and 16 healthy donors and was compared to that of two recombinant reference proteins from L. interrogans. This analysis revealed two peptides (SIR16-D1 and SIR16-H1) from the local isolates with good accuracy for the detection of acute leptospirosis (area under the ROC curve: 0.86 and 0.78, respectively; sensitivity: 0.88 and 0.94; specificity: 0.81 and 0.69), which was close to that of the reference proteins LipL32 and Loa22 (area under the ROC curve: 0.91 and 0.80; sensitivity: 0.94 and 0.81; specificity: 0.75 and 0.75). Conclusions/Significance This analysis lends further support for using ORFeome phage display to identify pathogen-associated immunogenic peptides, and it suggests that this technique holds promise for the development of peptide-based diagnostics for leptospirosis and, possibly, of vaccines against this pathogen.

  • ORFeome Phage Display.
    Methods of Molecular Biology, 2017
    Co-Authors: Jonas Zantow, Gustavo Marçal Schmidt Garcia Moreira, Stefan Dübel, Michael Hust
    Abstract:

    ORFeome phage display allows the efficient functional screening of entire proteomes or even metaproteomes to identify immunogenic proteins. For this purpose, randomly fragmented, whole genomes or metagenomes are cloned into a phage-display vector allowing positive selection for open reading frames (ORF) to improve the library quality. These libraries display all possible proteins encoded by a pathogen or a microbiome on the phage surface. Consequently, immunogenic proteins can be selected from these libraries using disease-related immunoglobulins from patient serum. ORFeome phage display in particular allows the identification of immunogenic proteins that are only expressed in the host-pathogen interaction but not in cultivation, as well as the detection of very low expressed and very small immunogens and immunogenic proteins of non-cultivable organisms. The identified immunogenic proteins are potential biomarkers for the development of diagnostic assays or vaccines. These articles will give an introduction to ORFeome phage-display technology and give detailed protocols to identify immunogenic proteins by phage display.

Yasushi Hiraoka - One of the best experts on this subject based on the ideXlab platform.