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.

Tobacco etch virus protease (TEVp) is a powerful enzymatic reagent for removing fusion tag. In this work, we constructed nine TEVp variants with introducing one to three mutations of C19S, C110S and C130S into the soluble TEVp variant, TEVp5M. Using the C-terminal green fluorescent protein (GFP) variant reporter, all constructs showed different solubility levels among four E. coli strains. The TEVp5M containing the C110S and/or C130S mutations in the hyperoxic strain showed the enhanced the cleavage activity. Addition of dithiothreitol to the cultural medium increased the activity of certain constructs produced in the BL21(DE3), contrary to the added hydrogen peroxide, due to cytoplasmic redox change measured by the redox sensitive GFP construct. The more cysteine residues in the purified TEVp5M were modified specifically than those in the other variants. All purified constructs showed similar specific activities in the presence of 5 mM dithiothreitol. In the buffer containing the compounds to aid disulfide bond formation of the refolded protein, the double mutant TEVp5MC110S/C130S exhibited the highest cleavage efficiency. This variant was efficient for removing the fusion tag after refolding of cellulose-binding module tagged disulfide-rich proteins including bovine enteropeptidase and maize peroxidase absorbed on the regenerated amorphous cellulose.

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