A protein-fragment complementation assay to quantify synthetic protein scaffold efficiency

Scaffolds are powerful tools in synthetic biology used for various applications, from increasing yield to optimizing signalling specificity. Protein scaffolds can be built by fusing peptide binding domains (PBD) and attaching the peptide they bind to enzymes, inducing spatial proximity. Only a few PBD-peptide combinations have been tested in this context, and no combination produced a high yield in yeast, an important chassis in biotechnology. Therefore, there is a need for more exploration of PBD-peptide pairs to be used in this model. Scaffold characterization is challenging because it is often dependent on a model pathway with an output that is difficult to measure quantitatively. Here, we use the dihydrofolate reductase protein-fragment complementation assay (DHFR PCA) to study scaffolding efficiency in yeast, which allows to couple scaffolding efficiency with growth rate. First, we characterize the strength of PBD-peptide interactions (PPI) and the binding availability of the PBDs and peptides. Then, we test different scaffold architectures and expression levels to quantify the simultaneous binding of peptide pairs to the scaffold. We show that PPI strength of the weakest binding PBD-peptide pair is critical for scaffolding efficiency and that PPI strength is limited by low binding availability of some domains and peptides in vivo. Also, we find that slight architectural variations and expression levels have a significant impact on scaffolding efficiency detected by DHFR PCA. Finally, we used DHFR PCA approaches to characterize novel PBD-peptide pairs and we identified pairs to expand the sequence toolbox for scaffold design in yeast through DHFR PCA easy-to-read signal.