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

Scaffolds are powerful tools in synthetic biology for various applications, from increasing yield to optimizing signalling specificity. Scaffolds can be built by fusing peptide binding domains (PBD) and attaching the peptide they bind to the 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 yeast. 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 a protein-fragment complementation assay (PCA) to study scaffold efficiency in yeast which allows to couple scaffold efficiency with growth rate. First, we characterize PBD-peptide interaction (PPI) strength and their binding availability. 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 PDB-peptide pair is critical for scaffold efficiency and that PPI strength is limited by low binding availability of some domains and peptides. Also, we find that slight architectural variations and expression levels have a significant impact on scaffold efficiency detected by DHFR PCA. In the future, DHFR PCA method will enable testing in a high throughput manner scaffold variants in yeast through its easy-to-read scaffolding efficiency signal.