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

The genes encoding feIFN-ωa and feIFN-ωb were subcloned into the prokaryotic soluble expression vector pCold-TF and then transformed into E. coli BL21 competent cells, thus generating the recombinant protein expressing E. coli strains pCold-feIFN-ωa/BL21 and pCold-feIFN-ωb/BL21. After inducing their expression with IPTG, the rfeIFN-ωa ( Figure 4a ) and rfeIFN-ωb (Figure 4b ) proteins fused with the trigger factor (TF) of approximately 57 kDa and were primarily expressed in their soluble forms. Subsequently, we optimized the expression conditions of the rfeIFN-ω proteins from the pCold-feIFN-ωa/BL21 and pCold-feIFN-ωb/BL21 E. coli strains. Our observations indicated that the optimal induction conditions for rfeIFN-ωa protein expression were an IPTG concentration of 1.0 mmol/L ( Figure 4c ) and an induction time of 8 h (Figure 4d) , and the optimal induction conditions for rfeIFN-ωb protein expression were an IPTG concentration of 0.8 mmol/L ( Figure 4e ) and an induction time of 10 h (Figure 4f ). After that, the fused proteins (rfeIFN-ωa/ωb+TF) were purified by His-tag Ni 2+ affinity column chromatography and subjected to cleavage using the 3C protease. The cleaved proteins were then purified by His-tag Ni 2+ affinity column chromatography once again, after which we collected the purified recombinant rfeIFN-ωa and rfeIFN-ωb proteins with molecular weights of approximately 21 kDa and 22 kDa, respectively ( Figure 4g ).