Tag 절단 protease인 TurboTEV (TEV Protease)는 His, GST 태그가 추가된 독특한 유전자 개변형 protease입니다. His 태그 단백질, GST 태그 단백질의 어느 발현계에서도 one step 정제가 가능하며, 4℃에서도 활성을 유지하기 때문에 온화한 조건에서의 절단 반응 및 정제가 가능합니다.
b. 인식과 절단 부위 ; Glu - Asn - Leu - Tyr - Phe - Gln ↓ Gly
c. 활성의 정의 ; 1unit는 인식 배열을 가진 태그 단백질 3μg를 30 ℃, 1 시간에 85 % 이상이 절단되는 효소량
d. 사용 조건 ; 온도 4-37 ℃, pH 6.5-8.5에서 사용 가능합니다.
e. 형상 ; 25 mM Tris - HCl (pH8.0) 50 mM NaCl, 1 mM TCEP 50 % Glycerol
A. Product detail
Accelagen's TEV protease, TurboTEV, contains an enhanced form of a catalytic fragment of the N1a protein of Tobacco
etch virus (TEV), a cysteine protease that recognizes the cleavage site of Glu-Asn-Leu-Tyr-Phe-Gln-Gly/Ser and cleaves
between Gln and Gly/Ser. TurboTEV Protease is a restriction grade protease that has a robust activity at 4 ℃ with high
specificity and great stability. It does not require any special buffer for its activity and can be used in a buffer most
suitable for the target protein. TurboTEV Protease is a 52 kDa protein with both GST and His tags so it can be removed by
either Ni-chelating or Glutathione (GSH) resin.
4. Specific activity
> 10 Units/ug. 1 Unit of TurboTEV Protease cleaves >85% of 3 ug of control substrate in 1 hour at 30℃.
Store at -20°C. Shipped in gel packs.
2 mg/ml in 25 mM Tris-HCl, pH8.0, 50 mM NaCl, 1 mM TCEP, 50% glycerol
2. Protease Activity
A 49 kDa GST-fusion protein (C) at 1 mg/ml is incubated with TurboTEV or TEV Protease at a ratio of (1) 1:50,
(2) 1:100, (3) 1:200 (w/w) in a buffer of 25 mM Tris-HCl, pH8.0, 150 mM NaCl, 14 mM b-mercaptoethanol at 4°C for
16 hours. The cleaved products are 27 kDa and 22 kDa. 'TEV' is a competitors’ TEV Protease product.
a. Make fresh cold Dialysis Buffer. Dialysis Buffer should be a buffer in which the target protein is soluble. There should be
no protease inhibitor in the Dialysis Buffer. The Dialysis Buffer should be compatible with downstream purification
processes, e.g. minimal amount of EDTA or DTT if Ni column will be used to remove the cleaved His-tag.
Here is an example of Dialysis Buffer. 25 mM Tris-HCl, pH 8.0, 150 - 500 mM NaCl, 14 mM b-mercaptoethanol
TurboTEV has the same activity in 150 mM NaCl or 500 mM NaCl and 400 mM imidazole.
b. Dilute the target protein pool 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 Uncut sample for analysis. EDTA may be added to 0.5 mM final concentration if
the target protein pool is eluted from Ni column and EDTA is compatible with the target protein.
c. Add TurboTEV Protease at a Protease:target protein ratio of 1:100 (w/w) or 10,000 unit (1 mg) TurboTEV Protease to
100 mg of target protein. There is no need to calculate the molar ratio. TurboTEV Protease can be added directly to the
target protein. There is no need to change buffer or dilute TurboTEV Protease. The optimal ratio should be
determined empirically. A Protease-to-target protein ratio (w/w) of 1:50 to 1:200 should work for most target proteins.
d. Dialyze against the Dialysis Buffer at 4 ℃ overnight (about 16 hrs). Dialysis is to remove imidazole or glutathione if Ni
or glutathione column is used to remove the cleaved tag or TurboTEV Protease after cleavage. If desired, the target
protein pool can be buffer exchanged first before TurboTEV cleavage.
2. Removal of TurboTEV Protease
a. The dialyzed target protein and TurboTEV Protease mixture can be applied directly to affinity columns if compatible
Dialysis Buffer is used. For His-tagged protein, use IMAC to remove the cleaved His-tag and TurboTEV Protease.
For GST-tagged protein, use glutathione column to remove the cleaved GST-tag and TurboTEV Protease.
b. If desired, analyze samples using SDS-PAGE analysis. The difference between the tagged and cleaved target protein
may be too small to detect by SDS-PAGE. The cleaved His-tag sometimes can be seen at the bottom of the gel.