A Sensitive, Semi-Quantitative Mammalian Two-Hybrid Assay

Abstract

Protein-protein interactions critically determine the function of a protein within the cell. Several methods have been developed for the analysis of protein interactions, including two-hybrid assays in yeast and mammals. Mammalian two-hybrid systems provide the ideal physiological environment to study the interactions of mammalian proteins; however, these approaches are limited in sensitivity and their ability to quantify interaction strength. Here, we present an inducible mammalian two-hybrid (iM2H) system using the small-molecule dimerizer rapalog for recruitment of multiple transactivation domains into the M2H system. This inducibility, combined with additional improvements of the iM2H components, results in an up to 100-fold increase in sensitivity compared with conventional M2H approaches. In addition, we include a number of reference interactions in our iM2H approach, which enable semiquantitative assessment of protein interactions. Using Groucho/Tle proteins and their binding partners, we demonstrate the applicability of our iM2H to established protein networks. Finally, to test the applicability of our system for drug screening, the interference of a small-molecule inhibitor on a known protein-protein interaction was tested, and the particular advantages of the internal reference interactions were shown.

A Dominant Negative Antisense Approach Targeting β-Catenin

“…In order to investigate the effects of the β-catenin truncations on its interactions, we performed a mammalian two-hybrid analysis [ 39 ]. We selected blocking of two SD regions for the analysis; in addition to the SD of exon 13 (aa 654–694) we used that of exon 6 (aa 248–314) as a control, which leads to truncations lacking the majority of the armadillo repeats.…”

“…The four truncated β-catenin versions served as prey in the mammalian two-hybrid assay and were N-terminally fused to the p65 transactivation domain. Tcf3, Lef1, cadherin and α-catenin were used as baits, fused to the DNA binding domain ZFHD [ 39 ].…”

“…Transfection was performed using TurboFect (Thermo Scientific) transfection reagent following instructions of the manufacturer. Measurement was performed as described [ 39 ]. For the mammalian two-hybrid experiments, 80 ng reporter plasmid (plucF24ZF), 2 ng reference (pMcGlucS) and 2 ng bait construct (see Table S1) were co-transfected per well with 22 ng truncated β-catenin constructs (prey) into HEK293 T-REx cells.…”

A dual luciferase assay for evaluation of skin sensitizing potential of medical devices

“…For optimization of the ARE reporter (ARE sequence, multimerization of ARE binding site, reporter gene) we used transient transfection experiments in HEK293 cells. The pMlucM reporter plasmids were derived from pMlucF backbone [33] by changing the minimal Fos promoter to the artificial minimal promoter (TAT AAA ATT CTC ATT CAG CCG ATA CCG TCT CAC TCT ). Four different ARE binding sites were inserted with oligo cloning into the multiple cloning site and mulitmerized up to 12 times.…”

“…Clones were tested for induction with 100 µM benzylideneacetone and selected for low basal NlucP levels and high induction rate. For more information on stable cell lines see S1 Table. Luciferase and viability measurement For all experiments except for Table 1, dual luciferase and resazurin measurements were performed as described previously [33]. For single luciferase measurement, only two dispensers (1 × substrate, 1 × H 2 SO 4 ) were used, and luciferase activity was measured directly after substrate dispensing.…”