Abstract
Tobacco etch virus (TEV) protease is a 27‐kDa catalytic domain of the polyprotein nuclear inclusion a (NIa) in TEV, which recognizes the specific amino acid sequence ENLYFQG/S and cleaves between Q and G/S. Despite its substrate specificity, its use is limited by its autoinactivation through self‐cleavage and poor solubility during purification. It was previously reported that T17S/N68D/I77V mutations improve the solubility and yield of TEV protease and S219 mutations provide protection against self‐cleavage. In this study, we isolated TEV proteases with S219N and S219V mutations in the background of T17S, N68D, and I77V without the inclusion body, and measured their enzyme kinetics. The k cat of two isolated S219N and S219V mutants in the background of T17S, N68D, and I77V mutations was highly increased compared to that of the control, and S219N was twofold faster than S219V without K m change. This result indicates that combination of these mutations can further enhance TEV activity.
Highlights
Tobacco etch virus (TEV) protease is a 27-kDa catalytic domain of the polyprotein nuclear inclusion a (NIa) in TEV, which recognizes the specific amino acid sequence ENLYFQG/S and cleaves between Q and G/S
The expression and purity of S219N and S219V mutant TEV proteases with the background of T17S/ N68D/I77V mutations were confirmed on SDS/PAGE via a single band with the expected molecular weight of 27 kDa (Fig. 2)
The activities of isolated T17S/N68D/I77V+S219V mutant TEV protease and T17S/N68D/I77V+S219N mutant protease were measured via the appearance of the cleaved substrate on SDS/PAGE and fluorescence substrates
Summary
Tobacco etch virus (TEV) protease is a 27-kDa catalytic domain of the polyprotein nuclear inclusion a (NIa) in TEV, which recognizes the specific amino acid sequence ENLYFQG/S and cleaves between Q and G/S. The catalytic triad residues of the active site are His, Asp, and Cys151, and are located at the interior of the domain (Fig. 1) These residues recognize the amino acid sequence ENLYFQG/S on a target protein and cleave the peptide bond between Q and G/S [6-9]. The T17S/N68D/ I77V mutant is more stable than the wild-type protease because the mutations result in more rigid secondary structure elements, such as helices and sheets [15] Another limitation is that the wild-type TEV protease truncates itself during production and purification, decreasing its activity [16].
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