An Optimized Strategy for Random Peptide Library Construction Facilitates Screening of HBc-Binding Peptides via Yeast...

Peptides have been widely used in therapeutic, diagnostic, and biotechnological applications due to their high affinity, specificity, and reduced immunogenicity compared to proteins, as well as superior potency, selectivity, and lower off-target toxicity compared to small molecules. Hepatitis B virus core protein (HBc), a component of the nucleocapsid of hepatitis B virus (HBV), has emerged as a promising target for the creation of new antiviral therapies. Here, we employed yeast surface display (YSD) combined with a rationally designed "3 + 4" peptide library to screen for high-affinity HBc-binding peptides. This library features segmented randomization (3 random amino acids + Gly-Ser (GS) + 4 random amino acids) to enhance combinatorial diversity while maintaining structural flexibility. Leveraging Golden Gate assembly, we achieved efficient and seamless library construction, enabling high-throughput screening via fluorescence-activated cell sorting (FACS). Three candidate peptides demonstrated strong binding to HBc, as validated by flow cytometry, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), split sfGFP complementation assay, and competitive magnetic bead pulldown assays. These peptides exhibit potential as lead compounds for developing HBc-targeting antivirals or diagnostic tools. Our study highlights the utility of YSD for functional peptide discovery and provides a framework for targeting complex viral proteins through combinatorial library design.