A FRET-based screening method to detect potential inhibitors of the binding of CNNM3 to PRL2.

ABSTRACT: The cyclin M (CNNM) family of Mg²⁺ transporters is reported to promote tumour progression by binding to phosphatase of regenerating liver (PRL) proteins. Here, we established an assay for detection of the binding between the cystathionine-beta-synthase (CBS) domain of human CNNM3 (a region responsible for PRL binding) and human PRL2 using fluorescence resonance energy transfer (FRET) techniques. By fusing YPet to the C-terminus of the CNNM3 CBS domain and CyPet to the N-terminus of PRL2, we performed a FRET-based binding assay with purified proteins in multiwell plates and successfully detected the changes in fluorescence intensity derived from FRET with a reasonable K_d. We then confirmed that the addition of non-YPet-tagged CNNM3 and non-CyPet-tagged PRL proteins inh

SUBMITTER: Cai F 

PROVIDER: S-EPMC7393355 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature