Construction, Investigation and Application of TEV Protease Variants with Improved Oxidative Stability

Abstract

Tobacco etch virus protease (TEVp) is a useful tool for removing fusion tags, but wild-type TEVp is less stable under oxidized redox state. In this work, we introduced and combined C19S, C110S and C130S into TEVp variants containing T17S, L56V, N68D, I77V and S135G to improve protein solubility, and S219V to inhibit self-proteolysis. The solubility and cleavage activity of the constructed variants in Escherichia 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 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 enhanced yields were released from the regenerated amorphous cellulose via affinity absorption of the cellulose-binding module as the affinity tag.

Extracellular production, purification and tag-removal of the N-terminally annexin- and ELP-tagged fluorescent proteins

“…The reason was attributed to that the cysteine residues in one TEVp variant molecule were linked to another molecule via the mis-matched disul de bonds, and C151 was responsible for catalysis. Mutation of the cysteine residues increases the TEVp oxidative stability [30]. In contrast, three TEVp variants with C110 and/or C130S mutations exhibited the relatively high cleavage activity (Fig.…”

“…In each fusion protein, the TEVp recognition sequence (ENLYFQG↓S, tevS) was incorporated. The plasmids for expressing the mutated TEVp including TEVp1 (S219V), TEVp2 (L56V/S135G/S219V), TEVp3 (T17S/N68D/I77V/S219V), TEVp4 (T17S/L56V/N68D/I77V/S135G/ S219V), and TEVp5 (T17S/L56V/N68D/E106G/I77V/S135G/ S219V), TEVp6 (C151A in TEVp4), TEVp7 (C110S in TEVp4) TEVp8 (C130S in TEVp4), TEVp9 (C110S and C130S in TEVp4), the GST-EmGFP as a TEVp substrate were constructed in our laboratory [29,30]. The bacteriophage ΦX174 gene E encoding the lytic protein (LyE) was inserted into the pBAD33.1 plasmid to yield pBAD-LyE for controlling the gene expression under the control of the P BAD promoter [31].…”

“…The TEVp cleavage activity was analyzed. The GST-EmGFP in crude extract, and the puri ed TEVp variant fusion was mixed with mass ratio of 10: 1 to react at 30°C for 1 h. The mixture was incubated with SDS-PAGE loading buffer at 40°C for 30 min, and subjected to SDS-PAGE analysis for in-gel uorescence display [30]. The EmGFP with the N-terminal cellulose-binding module (CBM) tag was immobilized on the regenerated amorphous cellulose with buffer A or washed with buffer A containing 5 mM dithiothreitol (DTT).…”

Rational Design of Disulfide Bridges in BbPETaseCD for Enhancing the Enzymatic Performance in PET Degradation

“…Furthermore, not all disulfide bridges of the BbPETase CD formed upon expression, which could be attributed to the E. coli Rosetta gami-B (DE3) strain. Expression of proteins with disulfide bridges by the E. coli Rosetta gami-B (DE3) strain may be not very effective [25]. Therefore, it may adversely affect the correct formation of disulfide bridges in BbPETase CD and its variants.…”

Between Protein Fold and Nucleophile Identity: Multiscale Modeling of the TEV Protease Enzyme–Substrate Complex

“…With the biotechnological use of an enzyme comes a need to improve or adjust its properties. Variants with improved solubility and yield, , reduced self-cleavage, and improved oxidative stability as well as immobilization strategies were reported . However, the biggest interest lies in the improvement of TEVp catalytic activity, since the wild type is slow compared to other widely used proteases that commonly implement serine as a nucleophile.…”