The Dihydrofolate Reductase Protein-Fragment Complementation Assay: A Survival-Selection Assay for Large-Scale Analysis of Protein-Protein Interactions.
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.
Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
Stephen W
SW
Michnick
Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100 Israel.
Emmanuel D
ED
Levy
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.
Christian R
CR
Landry
Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
Jacqueline
J
Kowarzyk
Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada.|Department of Medical Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
Vincent
V
Messier
D000818:Animals; D017930:Genes, Reporter; D005820:Genetic Testing / Q000379:methods*; D051379:Mice; D050296:Microbial Viability; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development*; D012641:Selection, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*
D013762:Tetrahydrofolate Dehydrogenase