Tobacco etch virus (TEV) protease with multiple mutations to improve solubility and reduce self‐cleavage exhibits enhanced enzymatic activity

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 kcat 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 Km change. This result indicates that combination of these mutations can further enhance TEV activity.

YESS 2.0, a Tunable Platform for Enzyme Evolution, Yields Highly Active TEV Protease Variants

“…Residue 219 is part of the flexible Cterminal loop positioned below the catalytic core and plays a critical role in regulating TEV autolysis and substrate binding. 43,44 Variant S6 reverted to the wild-type sequence and showed autolysis after purification as expected (Supplemental Figure 5B). Importantly, the respective mutations of S219 to Arg and Lys in variants S7 and S8 are shown for the first time to increase the catalytic efficiency of TEV while preventing autolysis (Supplemental Figure 5A).…”

“…On the one hand, S219P and S219E prevent TEV autolysis but occur at the detriment of k cat and K M , respectively . On the other hand, mutating S219 to Val, Asn, Lys, or Arg also prevents autolysis, yet, in addition, they result in increased catalytic activity , (Figure B). Whether residue 219 has greater impacts on K M or k cat may not be straightforward and may depend on the context of other mutations present in TEV.…”

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

“…Incubation of the purified M6 allowed the target proteins to be released from the affinity matrix. So far, different TEVp variants have been created for improving protein folding, solubility, thermostability, and catalytic activity [ 2 , 12 , 19 - 21 , 35 ]. Our work expands the TEVp toolkit, and the M6 variant is used for generating refolded tag-free bEK and mPex with biotechnological and medical values [ 24 , 25 ].…”

Steering towards success in stormy times: FEBS Open Bio in 2021

“…This article was the subject of our first commentary article [2], and we hope to commission commentaries to accompany other papers of especial significance in the future. Other exciting work across the molecular and cellular life sciences published in FEBS Open Bio this year includes a report of alterations in neuronal development in a mouse model of Timothy syndrome [3], the crystal structure of botulinum neurotoxin subtype A3 cell‐binding domain in complex with the ganglioside GD1a [4], engineered variants of tobacco etch virus protease with enhanced enzymatic activity [5], the discovery that autologous apoptotic neutrophils inhibit inflammatory secretion by dendritic cells [6] and the use of fluorescence microscopy to reveal cooperative binding of cardiac troponin and tropomyosin to filamentous actin [7]. These are but a few of the fascinating studies published this year in the journal.…”