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HRV3C Protease

GWB-15AECB

45-005-155001

1 mg (1000 units)
$255.00
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Description

Product Name HRV3C Protease.
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. HRV3C Protease is a restriction grade recombinant form of the HRV3C protease that has robust activity at 4°C with high specific activity 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. HRV3C Protease is a 47 kDa protein with both GST and His tags so it can be easily removed by either Ni-chelating or Glutathione (GSH) resin along with the cleaved tag. The activity of HRV3C Protease is tested using a control target protein. One (1) μg of HRV3C Protease has at least 1 unit activity conventionally used by other suppliers (unit of HRV3C protease cleaves >95% of 100μg of control target protein at 4°C for 16 hours). No non-specific activity has been observed under the same condition with HRV3C Protease to control target protein ratio of 1:10. Prolonged incubation (several days) under the same condition does not show any non-specific cleavage.
Protein Type Protease.
Source E. coli.
Specific Activity > 1 Units/mg. 1 Units of HRV3C Protease cleaves >95% of 100 mg of target protein at 4°C for 16 hours.
Cleavage in Solution
1. 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
HRV3C has the same activity in 150 mM NaCl or 500 mM NaCl and 400 mM imidazole.
2. 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.
3. Add HRV3C Protease at a Protease:target protein ratio of 1:100 (w/w) or 1,000 unit HRV3C Protease to 100 mg of target protein. There is no need to calculate the molar ratio. HRV3C Protease can be added directly to the target protein. There is no need to change buffer or dilute HRV3C 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.
4. Dialyze against the Dialysis Buffer at 4°C overnight (about 16 hrs). Dialysis is to remove imidazole or glutathione if Ni or glutathione column is used to remove the cleaved tag or HRV3C Protease after cleavage. If desired, the target protein pool can be buffer exchanged first before Rurbo3C cleavage.
Removal of HRV3C Protease
1. The dialyzed target protein and HRV3C 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 HRV3C Protease. For GST-tagged protein, use glutathione column to remove the cleaved GST-tag and HRV3C Protease.
2. 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.
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. HRV3C protease is a restriction grade recombinant form of the HRV3C protease that has robust activity at 4°C with high specific activity 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. Turby3C Protease is a 47 kDA protein with both GST and His tags so it can be easily removed by either Ni-chelating or Gluthathione (GSH) resin along with the cleaved tag.
Activity and Specificity The activity of HRV3C Protease is tested using a control target protein. One (1) ug of HRV3C Protease has at least 1 unit activity conventionally used by other suppliers (1 unit of HRV3C Protease cleaves ?95% of 100 ug of control target protein for at least 4°C for 16 hours). No non-specific activity has been observed under the same condition with HRV3C Protease to control target protein ratio of 1:10. Prolonged incubation (several days) under the same condition does not show any non-specific cleavage.
Storage and Stability Store HRV3C Protease at -20°C. HRV3C Protease is stable at room temperature for at least two weeks without loss of any activity. It retains full activity after incubation at 37C for one week.
Cleavage Condition It is recommended to use HRV3C Protease at a protease-to-target protein ratio of 1:100 (w.w) or 1 unit of HRV3C Protease to 100 ug of target protein in a buffer suitable for the target protein at 4°C overnight, with the target protein concentration at 1-2 mg/ml. In most cases, target proteins are completely cleaved with a protease-to-target protein ratio of 1:50 to 1:400 or 1 unit of HRV3C Proteaseto 50-400 ug of target protein (as shown in Figure 1). The efficiency of cleavage may vary due to the sequences around the cleavage site, the conformation and the solubility of the target protein. Due to its high specifity, more HRV3C Protease (at 1:10 ratio) or longer cleavage time (over a weekend) at higher temperature (37C) can be used to achieve high cleavage efficiency without non-specific cleavage of target proteins.
Removal of HRV3C Protease after Cleavage HRV3C Protease contains both GST and HIS tags. After cleavage of the target protein, HRV3C Protease can be easily removed along with the tags from the cleavage reaction by affinity chromatography on a Ni-chelating resin for His-tagged target protein or GSH resin for GST-tagged target protein.

Additional Information

Name HRV3C Protease
Related Product Names Purification, Cleavage of Protein His and GST Tag; HRV3C Protease Cleave Tagged Proteins; PreScission Site Specific Tags Removal HRV3C Protease
Datasheets / Downloads GWB-15AECB Datasheet
Concentration Lot Specific
Format This HRV3C Protease is in a storage buffer of 25 mM Tris-HCL, pH 8.0, 50 mM NaCl, 1 mM TCEP, and 50% glycerol. The storage buffer is compatible with Ni-chelating resin and GSH resin for on-column digestion.
Storage Store HRV3C Protease at -20C. HRV3C Protease is stable at room temperature for at least two weeks without loss of any activity. It retains full activity in storage buffer after incubation at 37C for one week.
Intended Use Research Use Only