Construction of an ASFV proteome library via multiple optimization strategies for high-throughput analysis

African swine fever virus (ASFV) is a large and structurally complex DNA virus encoding more than 160 proteins, including more than 68 structural proteins. A protein library covering recombinant ASFV proteins is fundamentally important for studies on protein function, antigenicity, vaccine development, and virus-host interactions. Here, to construct an ASFV protein library, we add a glutathione S-transferase (GST) tag at the N-terminus of each ASFV protein to facilitate solubilization and purification and express the recombinant proteins in the yeast host. By optimizing codons, expression vectors and strains and conditions of expression and purification, we achieve satisfactory protein yields for analytical applications and maximized access to the whole proteome of ASFV, with coverage of ca. 95%. Using the library, a protein chip is constructed and used to screen for interactions between ASFV and swine proteins ( e.g., IRF3, p65, and IκBα). The ASFV protein library lays the groundwork for understanding and combatting ASFV. The methods for constructing the library are instructive for generating other protein libraries for high-throughput applications.