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작성일 : 12-05-05
[Accelagen] Turbo3C(HRV 3C) Protease
GST 태그가 추가된 독특한 유전자 변형형 protease
 
Turbo3C (HRC 3C) Protease
 
A. Produc detail
 
1. Description
Human rhinovirus 3C protease (HRV3C Protease) is a cysteine protease that recognizes the cleavage site of Leu-Glu-Val-
Leu-Phe-Gln-Gly-Pro, commonly referred to as the PreScission Site. It cleaves between Gln and Gly. The recombinant
Turbo3C Protease is a restriction grade HRV3C protease that has robust activity at 4℃ and high specific activity. Turbo3C
Protease is a 47 kDa protein with both GST and His tags so it can be removed by either Ni chelating or Glutathione
(GSH) resin.
 
2. Protein type
    Protease
 
3. Source
    E. Coli
 
4. Specific activity
   > 1 Units/µg. 1 Units of Turbo3C (HRV3C) Protease cleaves >95% of 100 µg of target protein at 4℃ for 16 hours.
 
5. Storage/Handling
    Store at -20℃. Shipped in gel packs.
 
 
B. Data sheet
 
1. Formulation
   2 mg/ml in 50 mM Tris-HCl, pH8.0, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP, 50% glycerol
 
2. Protease Activity
 
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A 68 kDa GST-fusion protein (C) at 1 mg/ml is incubated with Turbo3C (HRV3C) Protease (*) at a ratio of (1) 1:50,
(2) 1:100, (3) 1:200, (4) 1:400 (w/w) in a buffer of 25 mM Tris-HCl, pH8.0, 150 mM NaCl, 14 mM ß-mercaptoethanol
at 4oC for 16 hours. The cleaved products are 42 kDa and 26 kDa
 
 
C. Protocol
 
1. Cleavage in Solution
 
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 ß-mercaptoethanol
 
Turbo3C has the same activity in 150 mM NaCl or 500 mM NaCl and 400 mM imidazole.
 
b. Dilute the 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 Turbo3C Protease at a Protease:target protein ratio of 1:100 (w/w) or 1,000 unit Turbo3C Protease to 100 mg
of target protein. There is no need to calculate the molar ratio. Turbo3C Protease can be added directly to the target
protein. There is no need to change buffer or dilute Turbo3C 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 Turbo3C Protease after cleavage. If desired, the target protein pool
can be buffer exchanged first before Rurbo3C cleavage.
 
2. Removal of Turbo3C Protease
 
a. The dialyzed target protein and Turbo3C 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 Turbo3C Protease.
For GST-tagged protein, use glutathione column to remove the cleaved GST-tag and Turbo3C 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.
 
 
Ordering information
 
Catalog No.
Product Name
Size / (Conc.)
 H0101S
Turbo3C(HRC 3C) Protease
1mg(1,000units) / (2mg/ml)
H0101M
10mg(10,000units) / (2mg/ml)
H0101B
Bulk
 
 
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