Designing a Self-Cleavable Construction Expressing Enterovirus 71 3C Protease

Abstract

3C protease is one of the most important proteases in enteroviruses, which is responsible for several cleavages of the viral polyprotein, leading to virus maturation. It has several harmful effects on host cells, such as the induction of caspase-mediated apoptosis and the cleavage of various proteins like P53, TATA-binding protein (TBP), histone H3, octamer-binding protein 1 (Oct-I) and DNA polymerase III. In this study, we aimed to construct a structure to express enterovirus 71 3C protease, consisting of a 3C protease sequence and two kinds of linkers including rigid and cleavable. As a result of cloning the structure into a pEGFP-N1 vector, two forms of constructions were expressed and verified by western blotting analysis including a 3C-Linker-Cleavage site (3C-L-CL) and a 3C-Linker (3C-L) without a cleavage site. In 3C-L-CL construction, 3C protease is released from fused GFP by cleave cleavage site. In 3C-L construction, 3C protease was expressed in fusion with GFP. In fact, one of the constructions produced two forms of 3C proteases, a free 3C and a 3C fused with GFP. This study shows that free 3C is preferred over the second form (3C with linker) because it is not connected to other peptides, and its protease activity is not affected. The designed structures could be used as useful tools for 3C protease expression and investigation of its interactions with cellular signaling pathways such as interferon pathway.