Combination of the mutations for improving activity of TEV protease in inclusion bodies.

Abstract

Tobacco etch virus protease (TEVp) is an enzymatic reagent to remove fusion tag, but additional purification steps are required for removing the TEVp after cleavage reaction is finished. Use of carrier-free and dependent TEVp immobilizates can eliminate protease contamination. In this work, we identified that, among the four constructed missense variants, the insoluble variant with the highest activity was correspondent with the soluble one tested formerly. The activities of the insoluble 15 codon variants were assayed and the variant with highest activity was selected. The K45F and/or E106G mutations have been reported on slightly improving protein stability of the wild-type TEVp, but only E106G mutation enhanced soluble production and activity of the selected TEVp variant, and it increased soluble amounts of two codon variants with the impaired folding. The decreased activity and use efficiency of the optimized TEVp variant in inclusion bodies was balanced by the determined high level production, lower leaking amounts of the protein, the enhanced resistance to the limited proteolysis mediated by protease K and trypsin, and the increased inhibition of auto-cleavage, as comparison to those of the immobilized soluble one. Thus, the TEVp construct is a potential alternate for simplifying protein purification protocols after tag-removal.