• Achieved ~95% coverage of the ASFV proteome by optimizing codon usage, expression vectors, yeast strains, and purification conditions.
• Constructed a protein chip from the library to screen virus-host protein interactions, identifying interactions with IRF3, p65, and IκBα.
• Demonstrated that yeast (Saccharomyces cerevisiae) is a suitable host for expressing a comprehensive viral protein library, overcoming challenges of solubility and yield.
• Provides a methodological framework applicable to constructing protein libraries for other large DNA viruses, facilitating functional genomics and vaccine development.