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 4oC for 16 hours. The cleaved products are 27 kDa and 22 kDa. 'TEV' is a competitors’ TEV Protease product.
Q) For your TEV digest reaction, the protocol states that the reaction is performed in 25 mMTris-HCl, pH 8.0, 150 mMNaCl and 14 mMβ-mercaptoethanol. I was wondering how important these buffer conditions are and whether the presence of any of the following may have an adverse affect on the cleavage efficiency of your TurboTEV?
a) 1mM DTT
b) 10% Glycerol
c) 20mM Histidine
d) 500 mMNaCl
e) 100mM Tris-HCl
f) Addition of β-mercaptoethanol
a) DTT of 1mM should be fine;
b) 10% Glycerol would not be recommended;
c) 20 mMHistidine would not be recommended;
d) 500mM NaClwould not be recommended;
e) 100mM Tris would not be recommended;
f) β-mercaptoethanol functions like DTT, so adding β-mercaptoethanol should not be a problem.
Q) Do we have to buffer exchange my protein prior to cleavage?
A) It may be necessary.
Step 1:Cleavage in Solution
- Make fresh cold Dialysis Buffer
When making fresh cold Dialysis Buffer, please be aware that:
a) The target proteinneeds to be soluble in the buffer
b) The buffer should NOT contain protease inhibitor
c) 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.
- Dilute the target protein pool
i. Dilute the target protein pool to 1-2 mg/ml with Dialysis Buffer.
Note:This is optional in case the target protein aggregates in Dialysis Buffer.
ii. 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.
- Add TurboTEV Protease
i. 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.
It is not required to change buffer or dilute TurboTEV Protease.
The optimal ratio should be determined by the user; however a Protease-to-target protein ratio (w/w) of 1:50 to 1:200 should work for most target proteins.
i. Dialyze against the Dialysis Buffer at 4oC overnight (about 16 hrs).This step 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.
Step 2:Removal of TurboTEV Protease
- Apply to columns
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.
- Optional:SDS-PAGE analysis
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.
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