LAUSR

Tobacco etch virus protease (TEVp) is a useful tool for removing fusion tag, but wild type TEVp is less stable under the oxidized redox state. In this work, we introduced and combined C19S, C110S and C130S into the TEVp variant containing T17S, L56V, N68D, I77V and S135G for improving protein solubility, and S219V for inhibiting self-proteolysis. The solubility and cleavage activity of the constructed variants in E. coli strains including BL21(DE3), BL21(DE3)pLys, Rossetta(DE3) and Origami(DE3) under the same induction conditions were analyzed and compared. The desirable soluble amounts, activity, and oxidative stability were identified to be reluctantly favored in the TEVp. Unlike C19S, C110S and C130S hardly impacted on decreasing protein solubility in the BL21(DE3), but they contributed to the improved tolerance to the oxidative redox state in vivo and in vitro. After two fusion proteins were cleaved by purified TEVp protein containing double mutations under the oxidized redox state, the refolded disulfide-rich bovine enterokinase catalytic domain or maize peroxidase with the enhanced yields was released from the regenerated amorphous cellulose via the affinity absorption of the cellulose-binding module as the affinity tag.