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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 \u201c3 + 4\u201d peptide library to screen for high\u2010affinity HBc\u2010binding peptides. This library features segmented randomization (3 random amino acids + Gly\u2010Ser (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\u2010throughput screening via fluorescence\u2010activated 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\u2010targeting 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.<\/jats:p>","DOI":"10.1002\/biot.70201","type":"journal-article","created":{"date-parts":[[2026,3,8]],"date-time":"2026-03-08T17:00:23Z","timestamp":1772989223000},"source":"Crossref","is-referenced-by-count":0,"title":["An Optimized Strategy for Random Peptide Library Construction Facilitates Screening of HBc\u2010Binding Peptides via Yeast Surface Display"],"prefix":"10.1002","volume":"21","author":[{"given":"Jin\u2010Lan","family":"Wang","sequence":"first","affiliation":[{"name":"Key Laboratory of Molecular Biology On Infectious Diseases Ministry of Education Chongqing Medical University  Chongqing China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yu\u2010Zhu","family":"Hu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Molecular Biology On Infectious Diseases Ministry of 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