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

Published inCold Spring Harbor protocols, vol. 2016, no. 11, p. 963-972
Publication date2016-11-01
First online date2016-11-01
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.

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Citation (ISO format)
MICHNICK, Stephen W. et al. The Dihydrofolate Reductase Protein-Fragment Complementation Assay: A Survival-Selection Assay for Large-Scale Analysis of Protein–Protein Interactions. In: Cold Spring Harbor protocols, 2016, vol. 2016, n° 11, p. 963–972. doi: 10.1101/pdb.prot090027
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ISSN of the journal1559-6095
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