Exploring the Mutations on Improving Oxidative Stability of Tobacco Etch Virus Protease for Tag-removal in Refolding of Two Disulfide-rich Proteins

Abstract

Tobacco etch virus protease (TEVp) is a useful tool for removing fusion tag, but wild type TEVp shows less oxidative stability, which limits its application under the oxidized redox state to facilitate disulfide bonds formation for refolding disulfide-bonded proteins. Previously, we combined six mutations into the TEVp to generate the TEVp5M for obviously increasing the protein solubility and decreasing the auto-cleavage. In this work, we introduced and combined C19S, C110S and C130S mutations into the TEVp5M to generate seven variants, analyzed protein solubility and the cleavage activity of the constructs in each of three E. coli strains including BL21(DE3), BL21(DE3)pLys, and Rossetta(DE3), and those of the optimized soluble variants in the oxidative cytoplasm of Origami(DE3) under the same induction conditions. The results suggested that desirable protein solubility, cleavage activity and oxidative stability are not combined. Unlike that of the C19S, introduction of the C110S and/or C130S less affected protein solubility but increased tolerance to the oxidative redox state. Use of the TEVp5MC110S/C130S variant, the refolded disulfide-rich bovine enteropeptidase or maize peroxidase was released via cleaving the sequence between the target protein and the cellulose-binding module bound to regenerated amorphous cellulose.