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T4455 Sigma

TEV Protease

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Properties

Related Categories Application Index, Biochemicals and Reagents, Enzymes, Inhibitors, and Substrates, Nanodisc Reagents for Membrane Protein Research, Proteases & Protein Sequencing,
recombinant   expressed in E. coli
description   Contains both a histidine tag and a GST tag
form   aqueous glycerol solution
activity   ≥10,000 units/mg protein
shipped in   wet ice
storage temp.   −20°C

Description

Biochem/physiol Actions

TEV protease has a strict 7 amino acid cleavage recognition sequence of Glu-Asn-Leu-Tyr-Phe-Gln↓Gly.

Features and Benefits

Optimally engineered and purified for enhanced stability and high specific activity over a broad temperature range.

General description

The tobacco etch virus (TEV) protease is a useful tool for the removal of fusion tags from recombinant proteins.

Preparation Note

Cleavage Protocol
Prepare fresh dialysis buffer. Dialysis buffer should be optimized for target protein solubility and contain no protease inhibitors. The dialysis buffer should also be compatible with downstream purification processes, e.g. minimal amount of EDTA or DTT if a HIS Select® column will be used to remove the cleaved His-tag.
Example of suitable dialysis buffer; 25 mM Tris-HCl, pH 8.0, 150 - 500 mM NaCl, 14 mM β-mercaptoethanol
This TEV protease has the same activity in 150 mM NaCl or 500 mM NaCl and 400 mM imidazole.
Dilute the target protein sample to 1-2 mg/ml with dialysis buffer. This is optional in case the target protein aggregates in dialysis buffer. Save a small aliquot as a control for PAGE analysis. EDTA may be added to 0.5 mM final concentration if the target protein will be eluted from the HIS Select column and EDTA is compatible with the target protein.
Add TEV protease at a protease to target protein ratio of 1:100 (w/w) or 10,000 unit (1 mg) TEV protease to 100 mg of target protein. There is no need to calculate the molar ratio. TEV protease can be added directly to the target protein. There is no need to change buffer or dilute TEV protease. The optimal ratio should be determined empirically. A Protease-to-target protein ratio (w/w) of 1:50 to 1:200 should provide an affective range for most target proteins.
Dialyze against the dialysis buffer at 4 °C ~ 16 hrs. Dialysis is intended to remove imidazole or glutathione if HIS Select® or glutathione affinity columns are used to remove the cleaved tag or TEV protease after cleavage.
Typically, 1 mg of TEV protease will cleave >90% of 100mg of a control protein at 4 °C in 16 hours.

Unit Definition

One unit of TEV protease cleaves >85% of 3 μg of control substrate in one hour at pH 8.0 at 30 °C.

Physical form

Supplied as a 2 mg/ml in 25 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM TCEP, and 50% glycerol

Application

Immobilized TEV protease on streptavidin-agarose is an efficient tool for fusion protein cleavage. TEV protease has also been used in a study to investigate proteolytic processing of nlrp1b for inflammasome activity.

Physical properties

A modified 52 kDa TEV protease construct containing both GST and His tags for easy removal using glutathione or His-Select® affinity media.

TEV protease is an engineered catalytic domain of the Tobacco Etch virus NIa protease. TEV protease is a highly specific cystein protease belonging to the C4 peptidase family.

Legal Information

HIS-Select is a registered trademark of Sigma-Aldrich Co. LLC

HiScreen is a trademark of GE Healthcare

Price and Availability

Safety & Documentation

Safety Information

RIDADR 
NONH for all modes of transport
WGK Germany 
2

Documents

Certificate of Analysis

Certificate of Origin

Protocols & Articles

Articles

Nanodisc Technology: A Revolutionary System for Study of Membrane Proteins

Membrane proteins are central to biological signaling pathways, regulating transfer of information and energy across cell membranes. They compose roughly 30% of the human proteome, but due to their k...
Carolyn Crankshaw, Product Specialist, Sigma® Life Science
Biofiles, Vol. 8, No. 20
Keywords: Cell disruption, Coagulation, Culture media, Detergents, Immobilization, Immunoprecipitation, Ligands, Nuclear magnetic resonance spectroscopy, Raman spectroscopy, Respiratory, Spectroscopy

Peer-Reviewed Papers
15

References

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