In vitro measurements of protein-protein interaction for the verification of the mammalian two hybrid system (trM2H)

Protein-Protein interactions are critical for cellular processes including gene regulation, cell signaling, and cell differentiation. Previous intracellular measurement systems have been developed to measure protein interactions, such as the yeast two-hybrid system and the commercial CheckMate Mammalian two-hybrid system, but these systems have substantial drawbacks. The yeast-two hybrid system may not give an accurate representation of mammalian protein interactions, and the CheckMate system has substantial background readings when testing proteins with Kd values over 1 μM. The Mammalian two-hybrid system (trM2H) developed by Zhang et al, provides accurate readings of protein-protein interactions with Kd values from 0.99 nM to 55 μM. To continue to verify the efficacy of the trM2H system, our team has purified S100 calcium-binding protein A7 (S100A7), also known as psoriasin, and performed a series of isothermal titration calorimetry studies to accurately determine the dissociation constant of the psoriasin homodimer. From our ITC data, the Western blot of purified psoriasin, and the previous fluorescent readings obtained with the trM2H, our group was unable to detect a homodimer Kd value close to those reported in previous literature