Development of a yeast protein fragment complementation assay (PCA) system using dihydrofolate reductase (DHFR) with specific additives.
A yeast protein fragment complementation assay (PCA) system based on dihydrofolate reductase (DHFR) is difficult to be operated because it is not as sensitive to trimethoprim (TMP) as the system using a prokaryotic microorganism. Here, the PCA system using DHFR, specific inhibitors, and a substrate in the yeast Saccharomyces cerevisiae was newly developed. As a model, the human oncoprotein Ras and the Ras-binding domain (RBD) of Raf-1 were individually and genetically fused to DHFR fragment, and each genetic construct was coexpressed under the control of the GAL1 promoter. An interaction between Ras and RBD could be evaluated on the basis of cell proliferation. To establish the experimental conditions for the yeast PCA system based on the DHFR reconstitution, we examined yeast host strains and the concentration of inhibitory additives to prevent endogenous DHFR activity, namely, TMP and sulfanilamide, and the substrate of DHFR, namely, folic acid. The transformant harboring wild-type Ras or its variants showed positive interaction signals, and the order of interactions for combination corresponded to the results of other in vitro assays. Moreover, combinatorial mutated Ras-binding domains were constructed, and the interaction of RBDs with Ras using this yeast PCA system was examined. As a result, various types of mutated clone for RBD were obtained. These demonstrations suggest that the yeast PCA system based on DHFR can be one of good, convenient, and inexpensive tools for investigating eukaryotic protein-protein interactions in vivo.
Shibasaki
Seiji
S
Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Minatojima, Chuo-ku, Kobe, 650-8530, Japan. seiji@huhs.ac.jp
Sakata
Kozue
K
Ishii
Jun
J
Kondo
Akihiko
A
Ueda
Mitsuyoshi
M
D004791:Enzyme Inhibitors / Q000494:pharmacology; D015723:Gene Library; D005818:Genetic Engineering; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D025941:Protein Interaction Mapping / Q000379:methods*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D014295:Trimethoprim / Q000494:pharmacology; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins