Detection of protein-protein interactions using a simple survival protein-fragment complementation assay based on the enzyme dihydrofolate reductase.
Biochemical 'pathways' are systems of dynamically assembling and disassembling protein complexes, and thus, much of modern biological research is concerned with how, when and where proteins interact with other proteins involved in biochemical processes. The demand for simple approaches to study protein-protein interactions, particularly on a large scale, has grown recently with the progress in genome projects, as the association of unknown with known gene products provides one crucial way of establishing the function of a gene. It was with this challenge in mind that our laboratory developed a simple survival protein-fragment complementation assay (PCA) based on the enzyme dihydrofolate reductase (DHFR). In the DHFR PCA strategy, two proteins of interest are fused to complementary fragments of DHFR. If the proteins of interest interact physically, the DHFR complementary fragments are brought together and fold into the native structure of the enzyme, reconstituting its activity, detectable by the survival of cells expressing the fusion proteins and growth in selective medium. Using the protocol described here, the survival selection can be completed in one to several days, depending on the cell type.
Remy
Ingrid
I
Département de Biochimie, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, Canada.
Campbell-Valois
F X
FX
Michnick
Stephen W
SW
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D051379:Mice; D008969:Molecular Sequence Data; D017510:Protein Folding; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000502:physiology
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase