The researcher successfully generated yeast vector libraries for the biosynthesis of ultra-diverse combinatorial cyclic oligopeptides (tetra- to octapeptides) with the general formula cyclo-NuX1X2X3–X7, where Nu is a nucleophilic residue (Cys, Ser, or Thr) and X represents any natural amino acid. Peptide-encoding sequences from the pre-constructed pEcoSICLOPPS-NuX1X2X3–X7 library were subcloned or PCR-amplified into the yeast centromeric vector pAG415 under the constitutive GAP promoter, enabling intracellular expression in Saccharomyces cerevisiae. Following transformation, library integrity and expression were assessed by SDS–PAGE and Western blot analysis, confirming production of the full-length precursor and its processed form, indicative of intein-mediated splicing and cyclic peptide formation.
In parallel, several clinically relevant membrane proteins (MPs), including PMP22, CLRN1, MC4R, GnRHR and V2R, were evaluated as potential reporters for the development of a yeast-based screening system. FLAG-tagged wild-type and mutant variants were expressed in Saccharomyces cerevisiae, with expression confirmed by SDS–PAGE and Western blot analysis. Although most MP variants were successfully produced, functional assessment using spheroplast-based immunofluorescence and flow cytometry analyses revealed limited differences in plasma membrane localisation between wild-type and mutant proteins, restricting their immediate applicability for high-throughput screening. In addition, fluorescence-based detection using anti-FLAG antibody labeling did not provide sufficient signal resolution for all tested MPs, while inhibition of proteasomal degradation with MG132 did not significantly improve fluorescence levels, indicating that proteasome-mediated degradation was not the primary limiting factor under the tested conditions.
To overcome these limitations, alternative strategies were explored to improve membrane targeting, detection sensitivity and assay robustness. A split fluorescent reporter system was developed, in which the target MP was fused to the mNeonGreen2(11) fragment, while the complementary mNeonGreen2(1–10) fragment was anchored to the plasma membrane. Validation of this system using the adenosine A2A receptor as a reporter protein demonstrated efficient membrane localisation and successful signal reconstitution. Furthermore, fusion of MPs to a deubiquitination (dUB) signal significantly improved plasma membrane localisation, highlighting a promising strategy for enhancing the performance and robustness of the yeast-based screening platform.
Finally, a new bacterial screening system available in the host laboratory was applied to identify cyclic peptides capable of rescuing the misfolding and ER retention phenotype of the disease-associated CLRN1(N48K) mutant. Following iterative fluorescence-activated cell sorting (FACS), progressive enrichment of fluorescent populations was observed over consecutive screening rounds, indicating potential successful selection of candidate rescuing molecules. Subsequent next-generation sequencing (NGS) analysis identified multiple peptide candidates for further functional evaluation regarding their ability to restore correct folding, membrane localisation, and trafficking of CLRN1(N48K).
