ORFeome cloning and systems biology: standardized mass production of the parts from the parts-list.
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.
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ABSTRACT: Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit's component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins.BMC Molecular Biology 08/2013; 14(1):18. · 2.06 Impact Factor
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ABSTRACT: Enterohemorrhagic E. coli (EHEC) manipulate their human host through at least 39 effector proteins which hijack host processes through direct protein-protein interactions (PPIs). To identify their protein targets in the host cells, we performed yeast two-hybrid screens, allowing us to find 48 high-confidence protein-protein interactions between 15 EHEC effectors and 47 human host proteins. In comparison to other bacteria and viruses we found that EHEC effectors bind more frequently to hub proteins as well as to proteins that participate in a higher number of protein complexes. The data set includes six new interactions that involve the translocated intimin receptor (TIR), namely HPCAL1, HPCAL4, NCALD, ARRB1, PDE6D, and STK16. We compared these TIR interactions in EHEC and enteropathogenic E. coli (EPEC) and found that five interactions were conserved. Notably, the conserved interactions included those of serine/threonine kinase 16 (STK16), hippocalcin-like 1 (HPCAL1) as well as neurocalcin-delta (NCALD). These proteins co-localize with the infection sites of EPEC. Furthermore, our results suggest putative functions of poorly characterized effectors (EspJ, EspY1). In particular, we observed that EspJ is connected to the microtubule system while EspY1 appears to be involved in apoptosis/cell cycle regulation.Scientific Reports 12/2014; 4:7531. · 5.08 Impact Factor
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ABSTRACT: Parallel analysis of translated open reading frames (ORFs) (PLATO) can be used for the unbiased discovery of interactions between full-length proteins encoded by a library of 'prey' ORFs and surface-immobilized 'bait' antibodies, polypeptides or small-molecular-weight compounds. PLATO uses ribosome display (RD) to link ORF-derived mRNA molecules to the proteins they encode, and recovered mRNA from affinity enrichment is subjected to analysis using massively parallel DNA sequencing. Compared with alternative in vitro methods, PLATO provides several advantages including library size and cost. A unique advantage of PLATO is that an alternative reverse transcription-quantitative PCR (RT-qPCR) protocol can be used to test binding of specific, individual proteins. To illustrate a typical experimental workflow, we demonstrate PLATO for the identification of the immune target of serum antibodies from patients with inclusion body myositis (IBM). Beginning with an ORFeome library in an RD vector, the protocol can produce samples for deep sequencing or RT-qPCR within 4 d.Nature Protocol 01/2014; 9(1):90-103. · 8.36 Impact Factor