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

Abstract

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.

1. Introduction

African swine fever (ASF) is a highly lethal infectious disease in domestic pigs. Since its outbreak in 1921, it has spread across continents several times and has caused considerable economic losses to the global pig industry [1,2]. The pathogen ASFV is a large DNA virus with a complex structure and a genome size of 170–193 kb. It encodes 150 to 167 proteins, including more than 68 structural proteins [3]. The functions of most of the encoded proteins have not been fully studied or understood [4]. The complexity of ASFV presents significant challenges for fundamental virological research and drug and vaccine development [5,6]. Obtaining recombinant proteins encoded by ASFV is essential for investigating their structures and biological functions [7,8]. Our goal is to obtain proteins encoded by the entire ASFV genome and construct an ASFV protein library, which will enable analyses of protein immunogenicity and functions for diagnostic marker identification and vaccine development from an omics perspective [9].

To achieve comprehensive and unbiased analysis, the library should ideally cover the entire viral proteome. There are documented strategies for using Saccharomyces cerevisiae as an expression host to express and purify the entire proteome of a species via high-throughput methods [10], as demonstrated with S. cerevisiae [11], Mycobacterium tuberculosis [12], Homo sapiens [13], and Zika and Dengue viruses [14]. However, the expression and purification characteristics can vary significantly among different proteins, making a protein library covering the whole proteome of a species, particularly for viruses with a large number of encoded proteins, a time-consuming, labor-intensive, and costly challenge [8,15]. Many parameters affect protein expression, solubility, and overall yield, including the plasmid copy number, the promoter, cultivation conditions (medium composition, dissolved oxygen, and shaking speed), the expression host, the time, and the type and concentration of the inducer [8,16]. Yeasts typically have smaller volumetric yields than bacteria do, but they excel in terms of secreted and membrane protein production compared with E. coli [17,18]. Therefore, the use of yeast as the expression host is conducive to obtaining a protein library with high genome coverage.

Here, we attempted to express all the coding products of the ASFV genome with Saccharomyces cerevisiae. The resulting recombinant proteins can be used for further studies on functionality, antigenicity, etc. By optimizing codon usage, switching expression vectors and hosts, and refining culture conditions and purification methods, we increased protein yields and genome coverage. The insights gained from this optimization process are valuable for the production of other recombinant protein libraries.