HRV 3C Protease
Ultra-pure HRV3C protease. Robust activity to cleave at the PreScission site with high specificity. His and GST-tags make it easy to remove from protein samples. 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 form of the HRV3C protease is a restriction grade protease that has robust activity at 4oC and high specific activity. This HRV3C 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.This product is available to US customers only.
Protein Type:
Protease
Source:
E. coli
Specific Activity:
> 1 Units/µg. 1 Units of Turbo3C (HRV3C) Protease cleaves >95% of 100 µg of target protein at 4oC for 16 hours.
Storage/Handling:
Store at -80oC.
Protease
Source:
E. coli
Specific Activity:
> 1 Units/µg. 1 Units of Turbo3C (HRV3C) Protease cleaves >95% of 100 µg of target protein at 4oC for 16 hours.
Storage/Handling:
Store at -80oC.
2 mg/ml in 50 mM Tris-HCl, pH8.0, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP, 50% glycerol
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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 b-mercaptoethanol at 4oC for 16 hours. The cleaved products are 42 kDa and 26 kDa |
Cleavage in Solution
- 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 Turbo3C has the same activity in 150 mM NaCl or 500 mM NaCl and 400 mM imidazole.
- 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.
- 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.
- Dialyze against the Dialysis Buffer at 4oC 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.
- 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.
- 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.
Vishwanatha K Back N, Mains RE, Eipper BA.. A Histidine-rich Linker Region in Peptidylglycine Alpha-Amidating Monooxygenase has the Properties of a pH-sensor. J Biol Chem. 2014 Mar 13.
Kurutihalli Vishwanatha,Nils Back,Richard E.Mains and Betty A.Eipper.A histidine-rich linker region in Peptidylglycine alpha-amidating Monoocygenase has the properties of a pH-sensor. The Journal of Biological Chemistry, 289, 12404-12420.