Publication

Purification of 10xHis-SuperTEV v1

2023

Abstract

SuperTEV is a mutated version of TEV (Tobacco Etch Protease) a cysteine protease widely used in labs as it is highly specific to a cleavage sequence that can be genetically encoded. Depending on the lab, it has been reported that the protease is unstable or purifies with low yields. Efforts have been made for many years (Tropea, 2009, Methods Mol Biol) to improve its solubility. Here we report our platform's efforts in generating a new TEV variant, called SuperTEV, that incorporates 9 mutations identified by different groups in recent years applied together at once. We find that the protein is easily produced and purified in large amounts and is functional.

The list of mutations on the canonical TEV protease are the following (with references that also identified the same mutations):

T17S (directed evolution, increased solubility and production) van den Berg et al., 2006 and Wei et al., 2012 L56V (rational design, improved solubility) Cabrita et al., 2007 and Wei et al., 2012 N68D (directed evolution, increased solubility and production) van den Berg et al., 2006 and Wei et al., 2012 I77V (directed evolution, increased solubility and production) van den Berg et al., 2006 and Wei et al., 2012 S135G (rational design, increased solubility) Cabrita et al., 2007 and Wei et al., 2012 I138T (increased catalytic activity; TEV3) Sanchez and Ting, 2019 S153N (increased catalytic activity; TEV3) Sanchez and Ting, 2019 T180A (increased catalytic activity; TEV3) Sanchez and Ting, 2019 S219V (inhibits autoproteolysis) Kapust, 2001

Official source

https://infoscience.epfl.ch/entities/publication/75cb509e-4ec8-45f5-8897-432818ac8b22

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2023

Abstract

The list of mutations on the canonical TEV protease are the following (with references that also identified the same mutations):

Official source

https://infoscience.epfl.ch/entities/publication/75cb509e-4ec8-45f5-8897-432818ac8b22

About this result

Related concepts (32)

Cysteine protease

Cysteine proteases, also known as thiol proteases, are hydrolase enzymes that degrade proteins. These proteases share a common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic triad or dyad. Discovered by Gopal Chunder Roy in 1873, the first cysteine protease to be isolated and characterized was papain, obtained from Carica papaya. Cysteine proteases are commonly encountered in fruits including the papaya, pineapple, fig and kiwifruit. The proportion of protease tends to be higher when the fruit is unripe.

Proteolysis

Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. Uncatalysed, the hydrolysis of peptide bonds is extremely slow, taking hundreds of years. Proteolysis is typically catalysed by cellular enzymes called proteases, but may also occur by intra-molecular digestion. Proteolysis in organisms serves many purposes; for example, digestive enzymes break down proteins in food to provide amino acids for the organism, while proteolytic processing of a polypeptide chain after its synthesis may be necessary for the production of an active protein.

Catalytic triad

A catalytic triad is a set of three coordinated amino acids that can be found in the active site of some enzymes. Catalytic triads are most commonly found in hydrolase and transferase enzymes (e.g. proteases, amidases, esterases, acylases, lipases and β-lactamases). An acid-base-nucleophile triad is a common motif for generating a nucleophilic residue for covalent catalysis. The residues form a charge-relay network to polarise and activate the nucleophile, which attacks the substrate, forming a covalent intermediate which is then hydrolysed to release the product and regenerate free enzyme.

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