T4455
TEV Protease
Synonym(s):
P1 protease, TEVp, Tobacco Etch Virus protease, rTEV
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About This Item
UNSPSC Code:
12352204
NACRES:
NA.54
Recommended Products
biological source
microbial (Tobacco Etch virus)
Quality Level
recombinant
expressed in E. coli
description
Contains both a histidine tag and a GST tag
form
solution
specific activity
≥3,000 units/mg protein
mol wt
27 kDa
technique(s)
protein purification: suitable
suitability
suitable for purification of HIS tagged recombinant proteins
application(s)
genomic analysis
shipped in
wet ice
storage temp.
−20°C
General description
TEV protease has (NIa) protease catalytic domain which corresponds to a molecular weight of 27 kDa. It is unique with high specificity and is active at low temperature.
The tobacco etch virus (TEV) protease is a useful tool for the removal of fusion tags from recombinant proteins.
Application
TEV Protease has been used in the removal of histidine tag from recombinant mitogen activated protein kinase 14 (MAPK14), Connexin43 (Cx43) c-terminal fragment and δ1-pyrroline-5-carboxylate reductase from Oryza sativa. It has also been used in the elution of purified proteasomes from human leukemia cell lines K562.
Biochem/physiol Actions
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.
The tobacco etch virus (TEV) protease undergoes autolysis, but mutants of TEV protease are resistant to autolysis. Bacterial expression of recombinant TEV protease leads to poor yields and less solubility. Use of green fluorescent protein fusion TEV protease overcomes these setbacks and finds application in structural genomics research.
TEV protease has a strict 7 amino acid cleavage recognition sequence of Glu-Asn-Leu-Tyr-Phe-Gln↓Gly/Ser.
Features and Benefits
Optimally engineered and purified for enhanced stability and high specific activity over a broad temperature range.
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.
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
aqueous glycerol solution
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.
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.
Legal Information
HIS-Select is a registered trademark of Merck KGaA, Darmstadt, Germany
HiScreen is a trademark of Cytiva
Storage Class Code
10 - Combustible liquids
WGK
WGK 2
Flash Point(F)
Not applicable
Flash Point(C)
Not applicable
Certificates of Analysis (COA)
Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.
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Connexin43 forms supramolecular complexes through non-overlapping binding sites for drebrin, tubulin, and ZO-1
Ambrosi C, et al.
Testing, 11(6), e0157073-e0157073 (2016)
Coronin 2A (CRN5) expression is associated with colorectal adenoma-adenocarcinoma sequence and oncogenic signalling
Rastetter RH, et al.
BMC Cancer, 15(1), 638-638 (2015)
Egor E Diakonov et al.
Biochemical and biophysical research communications, 508(2), 368-373 (2018-12-07)
The ubiquitin proteasome system is involved in the regulation of most basic intracellular processes, and deregulation of this system can results in certain kinds of human diseases. Proteolytic core this system, the 20S proteasome, has been found in physiological fluids
Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro
Nallamsetty S, et al.
Protein Expression and Purification, 38(1), 108-115 (2004)
A novel method for high-level production of TEV protease by superfolder GFP tag
Wu X, et al.
BioMed Research International, 2009 (2010)
Articles
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This page shows how to perform a purification of His-tagged membrane proteins.
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