A pipeline for systematic yeast 2-hybrid matricial screening in liquid culture.

2019 ◽  
Author(s):  
Monachello Dario ◽  
Guillaumot Damien ◽  
Lurin Claire
Keyword(s):  
High Throughput ◽  
Protein Interaction ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Operating Cost ◽  
Open Reading Frames ◽  
Critical Element ◽  
Interactome Mapping ◽  
Interaction Map ◽  
Split Ubiquitin

Abstract Physical interactions mediated by proteins are a critical element of biological systems, and the analysis of interaction partners can provide valuable hints about unknown functions of a protein. Two major classes of experimental approaches are used for protein interaction mapping: analysis of direct interactions using binary methods such as yeast two-hybrid (Y2H) or split ubiquitin, and analysis of protein complexes through affinity purification followed by mass spectrometry. Thanks to his flexibility to low- and high-throughput approaches and a low operating cost the Y2H assay is widely used for high-throughput interaction mapping experiments. Moreover, it has now been shown that high-throughput methods can produce highly reliable interactome datasets1 2 3 4. Notably, in 2011 a proteome-wide binary protein-protein interaction map of the plant Arabidopsis thaliana 5 (Arabidopsis Interactome Mapping project – AIM) was described using a high-throughput binary interactome mapping pipeline based on the Y2H system and using a collection of ~8,000 open reading frames (8k_space). Here we describe a liquid pipeline for a high-throughput binary protein–protein Y2H screen of a pool of 50 proteins used as baits against a collection of ~12,000 Arabidopsis proteins encoded by sequence-verified ORFs (12k_space)6 7.

scholarly journals Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes

2015 ◽  
Vol 118 ◽  
pp. 81-94 ◽  
Author(s):  
Jean-Philippe Lambert ◽  
Monika Tucholska ◽  
Christopher Go ◽  
James D.R. Knight ◽  
Anne-Claude Gingras
Keyword(s):  
Protein Complexes ◽  
Affinity Purification ◽  
Interactome Mapping

scholarly journals Biochemical Characterization of Protein Complexes from the Helicobacter pylori Protein Interaction Map

2004 ◽  
Vol 3 (8) ◽  
pp. 809-819 ◽  
Author(s):  
Laurent Terradot ◽  
Nathan Durnell ◽  
Min Li ◽  
Ming Li ◽  
Jeremiah Ory ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Protein Interaction ◽  
Biochemical Characterization ◽  
Protein Complexes ◽  
Interaction Map

scholarly journals Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

2008 ◽  
Vol 183 (2) ◽  
pp. 223-239 ◽  
Author(s):  
Laura Trinkle-Mulcahy ◽  
Séverine Boulon ◽  
Yun Wah Lam ◽  
Roby Urcia ◽  
François-Michel Boisvert ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Cell Biology ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Specific Protein ◽  
Protein Interaction Data ◽  
Quantitative Mass Spectrometry ◽  
Cell Extracts ◽  
Interaction Partners

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.

scholarly journals A tyrosine kinase protein interaction map reveals targetable EGFR network oncogenesis in lung cancer

2020 ◽  
Author(s):  
Swati Kaushik ◽  
Franziska Haderk ◽  
Xin Zhao ◽  
Hsien-Ming Hu ◽  
Khyati N. Shah ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Tyrosine Kinase ◽  
Protein Interaction ◽  
Tyrosine Kinases ◽  
Mapk Pathway ◽  
Affinity Purification ◽  
List Type ◽  
Wild Type ◽  
Interaction Map

SUMMARYSignaling networks balance the activities of many physically interacting proteins and perturbations to this network influence downstream signaling, potentially leading to oncogenic states. Using affinity purification-mass spectrometry we defined this network for all 90 human tyrosine kinases revealing 1,463 mostly novel interactions between these key cancer proteins and diverse molecular complexes. Modulation of interactor levels altered growth phenotypes associated with corresponding tyrosine kinase partners suggesting that tumors may alter the stoichiometries of interactors to maximize oncogenic signaling. We show that the levels of EGFR interactors delineates this form of network oncogenesis in 19% of EGFR wild-type lung cancer patients which were mostly otherwise oncogene negative, predicting sensitivity to EGFR inhibitors in vitro and in vivo. EGFR network oncogenesis occurs through mechanistically distinct network alleles often in cooperation with weak oncogenes in the MAPK pathway. Network oncogenesis may be a common and targetable convergent mechanism of oncogenic pathway activation in cancer.HIGHLIGHTSA human tyrosine kinome protein interaction map reveals novel physical and functional associations.Dependence on oncogenic tyrosine kinases is modulated through perturbation of their interactors.EGFR network oncogenesis in up to 19% of EGFR wild-type lung cancers is targetable.EGFR network oncogenesis cooperates with weak oncogenes in the MAPK pathway.

scholarly journals A novel recombinant DNA system for high efficiency affinity purification of proteins in Saccharomyces cerevisiae

2015 ◽  
Author(s):  
Brian H Carrick ◽  
Lixuan HaO ◽  
Philip J Smaldino ◽  
David R Engelke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Efficiency ◽  
Recombinant Dna ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Single Step ◽  
Open Reading Frames ◽  
Tev Protease ◽  
Number Of Factors ◽  
Endogenous Proteins

Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. The tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background in a single step. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent will allow isolation of rare or unstable protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or chromatin-associated assemblies.

scholarly journals Deciphering Protein Complexes and Protein Interaction Networks by Tandem Affinity Purification and Mass Spectrometry

2002 ◽  
Vol 1 (3) ◽  
pp. 204-212 ◽  
Author(s):  
Anna Shevchenko ◽  
Daniel Schaft ◽  
Assen Roguev ◽  
W. W. M. Pim Pijnappel ◽  
A. Francis Stewart ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Tandem Affinity Purification ◽  
Protein Interaction Networks ◽  
Interaction Networks

Protein–protein interactions

2010 ◽  
Vol 38 (4) ◽  
pp. 875-878 ◽  
Author(s):  
Mike P. Williamson ◽  
Michael J. Sutcliffe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Present Article ◽  
High Throughput ◽  
Protein Interactions ◽  
Metabolic Pathway ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Good Evidence ◽  
Protein Protein Interactions ◽  
Two Hybrid

In the present article, we describe the two standard high-throughput methods for identification of protein complexes: two-hybrid screens and TAP (tandem affinity purification) tagging. These methods have been used to characterize the interactome of Saccharomyces cerevisiae, showing that the majority of proteins are part of complexes, and that complexes typically consist of a core to which are bound ‘party’ and ‘dater’ proteins. Complexes typically are merely the sum of their parts. A particularly interesting type of complex is the metabolon, containing enzymes within the same metabolic pathway. There is reasonably good evidence that metabolons exist, but they have not been detected using high-thoughput assays, possibly because of their fragility.

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