RESUMO
The rapid development of new technologies for the high throughput (HT) study of proteins has increased the demand for comprehensive plasmid clone resources that support protein expression. These clones must be full-length, sequence-verified and in a flexible format. The generation of these resources requires automated pipelines supported by software management systems. Although the availability of clone resources is growing, current collections are either not complete or not fully sequence-verified. We report an automated pipeline, supported by several software applications that enabled the construction of the first comprehensive sequence-verified plasmid clone resource for more than 96% of protein coding sequences of the genome of F. tularensis, a highly virulent human pathogen and the causative agent of tularemia. This clone resource was applied to a HT protein purification pipeline successfully producing recombinant proteins for 72% of the genes. These methods and resources represent significant technological steps towards exploiting the genomic information of F. tularensis in discovery applications.
Assuntos
Proteínas de Bactérias/genética , DNA Complementar/genética , Francisella tularensis/genética , Genes/genética , Genoma Bacteriano , Fases de Leitura Aberta/genética , Tularemia/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Francisella tularensis/crescimento & desenvolvimento , Humanos , Tularemia/microbiologia , Tularemia/patologiaRESUMO
The availability of an annotated genome sequence for the yeast Saccharomyces cerevisiae has made possible the proteome-scale study of protein function and protein-protein interactions. These studies rely on availability of cloned open reading frame (ORF) collections that can be used for cell-free or cell-based protein expression. Several yeast ORF collections are available, but their use and data interpretation can be hindered by reliance on now out-of-date annotations, the inflexible presence of N- or C-terminal tags, and/or the unknown presence of mutations introduced during the cloning process. High-throughput biochemical and genetic analyses would benefit from a "gold standard" (fully sequence-verified, high-quality) ORF collection, which allows for high confidence in and reproducibility of experimental results. Here, we describe Yeast FLEXGene, a S. cerevisiae protein-coding clone collection that covers over 5000 predicted protein-coding sequences. The clone set covers 87% of the current S. cerevisiae genome annotation and includes full sequencing of each ORF insert. Availability of this collection makes possible a wide variety of studies from purified proteins to mutation suppression analysis, which should contribute to a global understanding of yeast protein function.
Assuntos
Genômica/métodos , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Composição de Bases , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA Fúngico/química , DNA Fúngico/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Fases de Leitura Aberta/genética , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNARESUMO
Functional proteomics approaches that comprehensively evaluate the biological activities of human cDNAs may provide novel insights into disease pathogenesis. To systematically investigate the functional activity of cDNAs that have been implicated in breast carcinogenesis, we generated a collection of cDNAs relevant to breast cancer, the Breast Cancer 1000 (BC1000), and conducted screens to identify proteins that induce phenotypic changes that resemble events which occur during tumor initiation and progression. Genes were selected for this set using bioinformatics and data mining tools that identify genes associated with breast cancer. Greater than 1000 cDNAs were assembled and sequence verified with high-throughput recombination-based cloning. To our knowledge, the BC1000 represents the first publicly available sequence-validated human disease gene collection. The functional activity of a subset of the BC1000 collection was evaluated in cell-based assays that monitor changes in cell proliferation, migration, and morphogenesis in MCF-10A mammary epithelial cells expressing a variant of ErbB2 that can be inducibly activated through dimerization. Using this approach, we identified many cDNAs, encoding diverse classes of cellular proteins, that displayed activity in one or more of the assays, thus providing insights into a large set of cellular proteins capable of inducing functional alterations associated with breast cancer development.
