High-Throughput Expression of <i>C. elegans</i> Proteins

Luan, Chi Hao; Qiu, Shaowei; Finley, James B.; Carson, M.; Gray, Randal; Huang, Wen-Ying; Johnson, David H.; Tsao, Jun; Reboul, Jérôme; Vaglio, Philippe; Hill, David E.; Vidal, Marc; DeLucas, L.; Luo, Ming

doi:10.1101/gr.2520504

Cited by 94 publications

(65 citation statements)

References 30 publications

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“…In addition to the protein solubility and stability challenges, the lack of eukaryotic posttranslational modifications and chaperones restrict the use of the Escherichia coli expression system in high-throughput proteomic studies. Only 48% of the 10,167 C. elegans ORFs were expressed in the E. coli expression system, and of these only 15% were soluble (13). Similar results were obtained for a small group of human proteins expressed in E. coli (14).…”

Section: Discussionsupporting

confidence: 64%

Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

Popescu

Popescu²,

Bachan³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

345

292

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Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/ CML proteins to regulate a wide range of targets and cellular activities.calmodulin-interacting proteins ͉ protein interaction network

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Section: Discussionsupporting

confidence: 64%

Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

Popescu

Popescu²,

Bachan³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

345

292

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“…Developing such "functional proteomic" approaches requires the design of high throughput and versatile experimental procedures (7,8). For the study of small G proteins, conventional filtration assays, although powerful at small scale, are not appropriate for systematic studies.…”

mentioning

confidence: 99%

Biochemical Clustering of Monomeric GTPases of the Ras Superfamily

Caruso

Jenna

Beaulne³

et al. 2005

Molecular & Cellular Proteomics

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To date phylogeny has been used to compare entire families of proteins based on their nucleotide or amino acid sequence. Here we developed a novel analytical platform allowing a systematic comparison of protein families based on their biochemical properties. This approach was validated on the Rho subfamily of GTPases. We used two high throughput methods, referred to as AlphaScreen Ras GTPases are small GTP-binding proteins involved in diverse cellular processes such as apoptosis, cell proliferation/differentiation, cytoskeleton reorganization, and membrane trafficking. These proteins cycle between an inactive GDP-bound form and an active GTP-bound form. Under their GTP-bound form their active conformation allows them to interact with effector molecules and to generate specific biological responses (1). Two intrinsic enzymatic activities regulate the balance between GTP-bound and GDP-bound conformations: 1) GDP/GTP exchange and 2) GTP hydrolysis activities (2). Whereas the intrinsic enzymatic activities have been characterized for a few of these proteins, such studies were carried out in non-systematic manners and by different research groups (3-6).The analysis of full genome sequences has allowed large scale biology experimental approaches consisting of systematic characterization of entire families of proteins instead of investigating them individually. Developing such "functional proteomic" approaches requires the design of high throughput and versatile experimental procedures (7,8). For the study of small G proteins, conventional filtration assays, although powerful at small scale, are not appropriate for systematic studies. Indeed this methodology has a low to very low throughput, is not versatile, and requires large amounts of reagents. To overcome these issues and characterize GTP-binding proteins of the Ras superfamily in a systematic manner, we developed a robust assay platform using two well adopted high throughput technologies, AlphaScreen TM (9) and FlashPlate® (10). AlphaScreen is a bead-based non-radioactive and homogenous proximity assay used to measure interaction between biological binding partners. The principle of this technology relies on the use of a Donor bead and an Acceptor bead that generate a light signal when brought into proximity (Ͻ200 nm). Upon laser excitation at 680 nm, the Donor beads, containing a photosensitizer, will generate short lived singlet oxygen that can diffuse only a short distance before returning to the ground state. The Acceptor beads, containing chemiluminescers and fluorophores, will react with this singlet oxygen and will emit an amplified light signal measurable at ϳ600 nm. AlphaScreen (PerkinElmer) provides highly versatile, sensitive, and homogeneous assays that allow us to perform studies at a higher throughput and at a lower cost.FlashPlates are white polystyrene microplates in which the interior of wells is coated with a thin layer of polystyrenebased scintillation reagent. The principle of this homogeneous radiometric technology is based on proximity betwe...

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“…This explains why the original primers did not generate any product in the first pass of the C. elegans ORFeome project . Despite the absent ORFs, Version 1.1 has already been used to express thousands of proteins for structural proteomics, proteomewide Y2H mapping, genome-wide RNAi analyses, and more (Chance et al 2002;Reboul et al 2003;Xu et al 2003;Li et al 2004;Luan et al 2004;Rual et al 2004a;Tewari et al 2004). Version 1.1 of the C. elegans ORFeome is developing into Version 2.1, in which single isolated clones corresponding to different splice products are captured from ORF minipools .…”

Section: Utility and Resource Compositionmentioning

confidence: 99%

ORFeome Cloning and Systems Biology: Standardized Mass Production of the Parts From the Parts-List

Brasch¹,

Hartley²,

Vidal³

2004

Genome Res.

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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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High-Throughput Expression of C. elegans Proteins

Cited by 94 publications

References 30 publications

Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

Biochemical Clustering of Monomeric GTPases of the Ras Superfamily

ORFeome Cloning and Systems Biology: Standardized Mass Production of the Parts From the Parts-List

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