ABSTRACT
T1R2/T1R3 belongs to G protein coupled receptors, which recognizes diverse natural and synthetic sweeteners. A novel class of positive allosteric modulators (PAMs) of T1R2/T1R3 was identified through high-throughput screening campaign. Comparing the structure of the potent compound with previously known PAM, we classified the structure of known PAM into three parts, defined as "head", "linker", and "tail". We then investigated the linker-tail structure. It was suggested by molecular docking models of T1R2/T1R3 that an amine that we introduced in the tail was the key for interaction with the receptor binding pocket. We thus synthesized various molecules and found unnatural tripeptide-PAMs, which potently enhance the sweetness of sucrose in sensory evaluation tests.
ABSTRACT
A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities.
