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The Dihydrofolate Reductase Protein-Fragment Complementation Assay: A Survival-Selection Assay for Large-Scale Analysis of Protein–Protein Interactions

Combining the Dihydrofolate Reductase Protein-Fragment Complementation Assay with Gene Deletions to Establish Genotype-to-Phenotype Maps of Protein Complexes and Interaction Networks.

Dissecting the Contingent Interactions of Protein Complexes with the Optimized Yeast Cytosine Deaminase Protein-Fragment Complementation Assay.

Real-Time Protein-Fragment Complementation Assays for Studying Temporal, Spatial, and Spatiotemporal Dynamics of Protein-Protein Interactions in Living Cells.

Micrococcal Nuclease Digestion of Schizosaccharomyces pombe Chromatin.

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Transcription Factor-DNA Binding Motifs in Saccharomyces cerevisiae: Tools and Resources.

Protein-Fragment Complementation Assays for Large-Scale Analysis, Functional Dissection, and Spatiotemporal Dynamic Studies of Protein-Protein Interactions in Living Cells.

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The Dihydrofolate Reductase Protein-Fragment Complementation Assay: A Survival-Selection Assay for Large-Scale Analysis of Protein–Protein Interactions

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Michnick, Stephen W. Levy, Emmanuel D. Landry, Christian R. Kowarzyk, Jacqueline Messier, Vincent
Description	Country affiliation: Canada Author Affiliation: Michnick SW ( Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada.); Levy ED ( Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100 Israel.); Landry CR ( Département de Biologie, Institut de Biologie Intégrative et des Systèmes, PROTEO-Québec Research Network on Protein Function, Structure and Engineering, Université Laval, Québec, Québec G1V 0A6, Canada.); Kowarzyk J ( Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada.); Messier V ( Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada.)
Abstract	Protein-fragment complementation assays (PCAs) can be used to study protein–protein interactions (PPIs) in any living cell, in vivo or in vitro, in any subcellular compartment or membranes. Here, we present a detailed protocol for performing and analyzing a high-throughput PCA screening to study PPIs in yeast, using dihydrofolate reductase (DHFR) as the reporter protein. The DHFR PCA is a simple survival-selection assay in which Saccharomyces cerevisiae DHFR (scDHFR) is inhibited by methotrexate, thus preventing nucleotide synthesis and causing arrest of cell division. Complementation of cells with a methotrexate-insensitive murine DHFR restores nucleotide synthesis, allowing cell proliferation. The methotrexate-resistant DHFR has two mutations (L22F and F31S) and is 10,000 times less sensitive to methotrexate than wild-type scDHFR, but retains full catalytic activity. The DHFR PCA is sensitive enough for PPIs to be detected for open reading frame (ORF)-PCA fragments expressed off of their endogenous promoters.
File Format	HTM / HTML
ISSN	19403402
Issue Number	11
Journal	Cold Spring Harbor Protocols
Volume Number	2016
e-ISSN	15596095
Language	English
Publisher	Cold Spring Harbor Laboratory Press
Publisher Date	2016-11-01
Publisher Place	United States
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Discipline Clinical Laboratory Techniques
Content Type	Text
Resource Type	Article
Subject	Biochemistry, Genetics and Molecular Biology

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Combining the Dihydrofolate Reductase Protein-Fragment Complementation Assay with Gene Deletions to Establish Genotype-to-Phenotype Maps of Protein Complexes and Interaction Networks.

Protein-Fragment Complementation Assays for Large-Scale Analysis, Functional Dissection, and Spatiotemporal Dynamic Studies of Protein-Protein Interactions in Living Cells.

Dissecting the Contingent Interactions of Protein Complexes with the Optimized Yeast Cytosine Deaminase Protein-Fragment Complementation Assay.

Protein-fragment complementation assays for large-scale analysis of protein-protein interactions

Real-Time Protein-Fragment Complementation Assays for Studying Temporal, Spatial, and Spatiotemporal Dynamics of Protein-Protein Interactions in Living Cells.

In Planta Visualization of Protein Interactions Using Bimolecular Fluorescence Complementation (BiFC).

Barcode fusion genetics-protein-fragment complementation assay (BFG-PCA): tools and resources that expand the potential for binary protein interaction discovery

Culturing large populations of Drosophila for protein biochemistry.

Multicolor bimolecular fluorescence complementation (BiFC) analysis of protein interactions with alternative partners.

The Dihydrofolate Reductase Protein-Fragment Complementation Assay: A Survival-Selection Assay for Large-Scale Analysis of Protein–Protein Interactions

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Protein-Fragment Complementation Assays for Large-Scale Analysis, Functional Dissection, and Spatiotemporal Dynamic Studies of Protein-Protein Interactions in Living Cells.

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The Dihydrofolate Reductase Protein-Fragment Complementation Assay: A Survival-Selection Assay for Large-Scale Analysis of Protein–Protein Interactions