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Updated: Mar 12, 2026
Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells
Published on: March 3, 2015
The Dihydrofolate Reductase Protein-Fragment Complementation Assay: A Survival-Selection Assay for Large-Scale
Stephen W Michnick1, Emmanuel D Levy2, Christian R Landry3
1Département 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.
