Grafting 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.
Fig: In vitro interaction of vGFP-scaffolded peptides with Mdm2 N-terminal domain (residues 6–125). Indicated purified proteins were (co)-incubated and the pulled down complexes analysed by SDS PAGE. Upper and lower arrows correspond to engineered vGFP proteins and interacting Mdm2, respectively. Experimental repeat is shown in Supplementary Fig.
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.
Chee, S.M.Q., Wongsantichon, J., Yi, L.S. et al. Functional display of bioactive peptides on the vGFP scaffold. Sci Rep 11, 10127 (2021). https://doi.org/10.1038/s41598-021-89421-y