Split Intein Enables Ultralarge Dual-Phage Display for Drug Discovery

Abstract Phage display, a powerful ligand discovery technology, is limited by library capacity, with practical diversity typically ∼109 variants. This bottleneck stems from each phage requiring individual host cell propagation, making ultralarge libraries (1012–1014) impractical due to massive cell/culture volume demands. We introduce X-phage, a dual-phage display system using split-intein-mediated ligation to combine two genetically distinct phages into a single functional entity. By randomly combining two small/medium sublibraries (106–107 each), a theoretical maximum diversity of up to 1.5 × 1013 is available in scFv libraries, using standard lab cell/culture volumes. The robustness of X-phage was validated by isolating low-nanomolar ligands against CD33 and TGFβR1; these were further engineered into bispecific engagers and immunomodulators for biotherapeutic applications. This “dual-phage single-phenotype” paradigm decouples library size from bacterial amplification, overcoming traditional capacity limits and enabling high-throughput functional molecule evolution. Thus, X-phage showcases how chemical approaches can advance biotechnology platforms.