Why should it be AG Scientific's HRV3C Protease?

Human rhinovirus 3C protease (HRV 3C 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 4°C and high specific activity and great stability.

Figure 1: HRV 3C cleavage

This enzyme does not require any special buffer for its activity and can be used in a buffer most suitable for the target protein. This HRV 3C 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 along with the cleaved tag.

Figure 2: HRV3C protease activity

HRV3C VS PRESCISSION PROTEASE™

Company GE HEALTHCARE AG SCIENTIFIC
Product PreScission Protease Human rhinovirus 3C protease, HRV 3C Protease
FORM: PreScission Protease is a genetically engineered fusion protein consisting of human rhinovirus 3C protease and GST. This protease was specifically designed to facilitate removal of the protease by allowing simultaneous protease immobilization and cleavage of GST fusion proteins produced from the pGEX-6P vectors pGEX-6P-1, pGEX-6P-2, and pGEX-6P-3 Recombinant form of the HRV3C protease is a restriction grade protease that has robust activity at low temperature (4°C) and high specific activity. This HRV 3C 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.
SUPPLIED AS: 2000 units/ml (833 to 1000 units/mg) in 50 mM
Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM EDTA, 1 mM DTT, and 20% glycerol
One mg of HRV 3C Protease in 500 ml at 2 mg/ml in a storage buffer of 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP, and 50% glycerol.

 

CLEAVAGE CONDITION: One unit will cleave > 90% of 100 ug of a test GST-fusion protein in 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.0 at 5°C for 16 h. It is recommended to test the HRV3C Protease cleavage with protease-to-target protein ratio of 1:100 (w/w) in the buffer suitable for the target protein at 4°C overnight, with the target protein concentration at or below 1 mg/ml. Under this condition, > 95% of target protein is cleaved in most cases. The efficiency of cleavage varies due to the sequences around the cleavage site, the conformation and the solubility of the target protein. Including reducing agent such as DTT or TCEP in the buffer helps the cleavage. Due to its high specificity, more protease can be used without non-specific cleavage of target proteins. No non-specific cleavage of control cleavage protein is observed at protease-to-control cleavage protein ratio of 1:10.
ACTIVITY & SPECIFICITY: 2000 units/ml (833 to 1000 units/mg) in 50 mM
Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM EDTA, 1 mM DTT, and 20% glycerol
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 (1 unit of HRV3C Protease cleaves >95% of 100 µg of control target protein at 4oC 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.

 

PROTEASE ACTIVITY: N/A 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 site: N/A
USA LIST SIZE 500 UNITS 1 mg (1000 UNITS)

 

 

*PreScission Protease is a registered trademark of GE HEALTHCARE Life Sciences Division

 

 

 

References

  1. Walker, P. A. et al. BIO/TECHNOLOGY 12, 601 (1994).
  2. Cordingley, M. G. et al. J. Biol. Chem. 265, 9062 (1990).