Comprehensive Biophysical Profiling Evidences Self-Oligomerization of Bacterially Expressed Pc Protein

Polycomb (Pc) protein is a core component of Polycomb Repressive Complex 1 (PRC1) and plays a central role in epigenetic gene silencing, particularly during embryo development. Despite extensive functional characterization, structural understanding of Pc remains limited due to its intrinsic disorder, low sequence complexity, and propensity for dynamic self-association. Here, we report the recombinant production, purification, and biophysical characterization of Drosophila melanogaster Pc constructs optimized for structural studies. Using solubility-enhancing fusion strategies, we obtained milligram quantities of stable MBP-tagged Pc protein suitable for downstream analyses. Sequence-based disorder prediction, circular dichroism, and solution-based techniques indicate that Pc is predominantly intrinsically disordered outside its conserved chromodomain, whereas it retains a defined secondary structure upon fusion and oligomerization. Thermal shift assays, Nano-Differential Scanning Fluorimetry (nanoDSF), dynamic light scattering (DLS), and Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) demonstrate strong buffer-dependent stability and concentration-dependent self-association into higher-order assemblies. Cryo-electron microscopy reveals pronounced conformational and oligomeric heterogeneity, which can be partially stabilized by gradient fixation. Complementary AlphaFold-based modeling supports a conditional folding mechanism in which Pc adopts more ordered architectures upon self-association. Together, our results establish a robust framework for the biophysical investigation of Pc protein and provide mechanistic insight into how intrinsic disorder, oligomerization, and self-association may contribute to Polycomb-mediated chromatin regulation.