Sharon Min Qi CheeJantana WongsantichonLau Sze YiBarindra SanaYuri FrosiRobert C. RobinsonFarid J. GhadessyVidyasirimedhi Institute of Science and TechnologyAgency for Science, Technology and Research, SingaporeOkayama UniversityMahidol University2022-08-042022-08-042021-12-01Scientific Reports. Vol.11, No.1 (2021)204523222-s2.0-85105792496https://repository.li.mahidol.ac.th/handle/20.500.14594/79248Grafting bioactive peptides into recipient protein scaffolds can often increase their activities by conferring enhanced stability and cellular longevity. Here, we describe use of vGFP as a novel scaffold to display peptides. vGFP comprises GFP fused to a bound high affinity Enhancer nanobody that potentiates its fluorescence. We show that peptides inserted into the linker region between GFP and the Enhancer are correctly displayed for on-target interaction, both in vitro and in live cells by pull-down, measurement of target inhibition and imaging analyses. This is further confirmed by structural studies highlighting the optimal display of a vGFP-displayed peptide bound to Mdm2, the key negative regulator of p53 that is often overexpressed in cancer. We also demonstrate a potential biosensing application of the vGFP scaffold by showing target-dependent modulation of intrinsic fluorescence. vGFP is relatively thermostable, well-expressed and inherently fluorescent. These properties make it a useful scaffold to add to the existing tool box for displaying peptides that can disrupt clinically relevant protein–protein interactions.Mahidol UniversityMultidisciplinaryArticleSCOPUS10.1038/s41598-021-89421-y