Cloning, Prokaryotic Soluble Expression, and Analysis of Antiviral Activity of Two Novel Feline IFN-? Proteins

Wang, Xiaona; Li, Fengsai; Han, Meijing; Jia, Shuo; Wang, Li; Qiao, Xinyuan; Jiang, Yanping; Cui, Wen; Tang, Lijie; Li, Yijing; Xu, Yi-Gang

Insect/baculovirus expression systems are one of the most effective eukaryotic expression systems for preparing feline IFNs. These expression systems are capable of making post-translational modifications, such as glycosylation, which allows proteins to fold correctly, thus producing highly active and stable IFNs [32] [33] [34] [35] [36] . However, the application of these systems is limited by drawbacks, such as low yield of IFN protein and complicated operation requirements [37] . Therefore, E. coli expression systems are still the most widely used prokaryotic expression system for protein production with their characteristics of simple procedures, large-scale production, and low cost [38] [39] [40] . Generally, recombinant IFN is produced by E. coli expression systems in an insoluble inclusion body form that has not been modified or folded correctly, resulting in the loss of its biological activity [41, 42] . To obtain a soluble and biologically active IFN protein, the inclusion bodies need to be denatured and then renatured, which is a time-consuming, laborious process [40] . In this study, we selected the prokaryotic soluble expression system pCold-TF to prepare recombinant feIFN-ωa and feIFN-ωb. The pCold-TF system is a highly efficient soluble expression system with a His-tagged TF that allows the expressed protein to be effectively modified, folded, and secreted into the cytoplasm [43, 44] . Following the construction of recombinant E. coli strains and induction by IPTG, the rfeIFN-ωa/ωb proteins were expressed in their soluble forms and analyzed via SDS-PAGE. We optimized expression conditions for the rfeIFN-ωa and rfeIFN-ωb proteins and purified them using His-tag Ni 2+ affinity column chromatography. Both proteins showed antiviral activity against VSV in microdose cytopathic effect inhibition assays using F81 cells. Our results provide a basis for further studies into the development of rfeIFN-ωa and rfeIFN-ωb as therapeutic agents for viral infections. Meanwhile, our data provide evidence that the pCold-TF expression system can be used to produce IFN with full bioactivity.