Selection of tobacco etch virus protease variants with enhanced oxidative stability for tag-removal in refolding of two disulfide-rich proteins

Abstract

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.

Ultrasound-assisted innovations in protein processing: review

“…Several factors influence the efficiency of the refolding process, including protein concentration, disulfide bond formation, chaperone proteins (such as small heat shock proteins and Hsp70 chaperones), micelle size and composition, and salt concentration. Increasing salt concentration up to 1 M NaCl can enhance the refolding process [75]. Additionally, synthetic nano chaperones with hydrophobic microdomains can stabilize denatured proteins and facilitate their refolding with high efficiency [76].…”