T4455

TEV Protease

• 동의어(들): P1 protease, TEVp, Tobacco Etch Virus protease, rTEV
• UNSPSC 코드: 12352204
• NACRES: NA.54

속성

• 생물학적 소스: microbial (Tobacco Etch virus)
• Quality Level: 200
• 재조합: expressed in E. coli
• 설명: Contains both a histidine tag and a GST tag
• 양식: solution
• 특이 활성도: ≥3,000 units/mg protein
• 분자량: 27 kDa
• 기술: protein purification: suitable
• 적합성: suitable for purification of HIS tagged recombinant proteins
• 응용 분야: genomic analysis
• 배송 상태: wet ice
• 저장 온도: −20°C

설명

일반 설명

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.

애플리케이션

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.

생화학적/생리학적 작용

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.

특징 및 장점

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

물리적 특성

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.

단위 정의

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

물리적 형태

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

제조 메모

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.

법적 정보

HIS-Select is a registered trademark of Merck KGaA, Darmstadt, Germany

HiScreen is a trademark of Cytiva

안전성 정보

• Storage Class Code: 10 - Combustible liquids
• WGK: WGK 2
• Flash Point (°F): Not applicable
• Flash Point (°C): Not applicable