|2000 U||Recombinant HRV 3C Protease (lyophilized from 50mM Tris, 150mM NaCl, 1mM EDTA, 1mM DTT, 0.04% Tween20, 8% trehalose, 8% mannitol)|
|100 μg||Cleavage Control Protein (lyophilized from sterile PBS, pH 7.4)|
|5 ml||10X HRV 3C Cleavage Buffer (1.5 M NaCl, 0.5 M Tris-HCl, pH 7.5)|
HRV 3C Protease encoded by human rhinovirus 14 is a highly purified recombinant cysteine protease with a His-tag. Recombinant HRV 3C Protease is a ~20KDa single-chain protein containing approximately 189 amino acids with calculated pI 8.46. HRV 3C protease folds into two anti-parallel six-stranded β-barrels and the site cleft is located at the junction of the two β-barrels domains. The enzyme requires neither metal nor cofactors for activity. It has been demonstrated that the enzyme exhibits highest activity around neutral pH at temperature ranging from 22 to 37℃, even retaining robust activity at 4℃. Thus, cleavage can be performed at low temperature to enhance the stability of the target protein. The catalytic activity is insensitive to organic solvents (up to 10%); however, it can be strongly stimulated by high concentration of anions such as sulfate.
The enzyme recognizes the cleavage site:
~22KDa on SDS-PAGE
Store HRV 3C Protease at –20°C. Store HRV 3C Cleavage Control Protein and Protease Cleavage Buffer at –20°C or 4°C.
Resuspend the enzyme powder with 1mL sterile water. Keep reconstituted enzyme at -20℃ in aliquots. Avoid repeated freeze-thaw cycles.
>90% by SDS-PAGE.
The purity of each lot is determined by SDS-PAGE. And the activity is ensured by cleavage test with a recombinant fusion protein for each lot. The solution of HRV 3C protease is filtered through 0.22μm sterile filter before package.
The high specificity of HRV 3C protease makes it an ideal tool for cleaving fusion proteins at definite cleavage sites. The fusion protein can be purified and cleaved by HRV 3C to obtain the target protein. The recombinant HRV 3C protease is easily removed by IMAC Ni-charged resin.
One unit of HRV 3C Protrease is defined as the amount of enzyme that will cleave>95% of 100μg HRV 3C cleavage control protein in 150mM NaCl, 50mM Tris-HCl pH 7.5, at 4℃ for 16h.
Incubation time: 16 hours or overnight Enzyme amount: 1:25~1: 100 (U/μg) Empirically, a HRV 3C protease: target protein ratio of 1:25~1: 100 (U/μg) at 4℃ for 16 hours is applicable for most fusion protein cleavage.
Due to various properties of fusion proteins, the ratio of HRV 3C protease: target protein, temperature, incubation time is recommended to be optimized for practical application. The following protocol is a simple example to estimate the appropriate amount of the enzyme.
1. Combine 100μg fusion protein, 10μl 10X HRV 3C protease Cleavage Buffer, HRV 3C protease of different volumes and sterile water to make a 100μl total reaction volume. A control sample without HRV 3C protease should be included to detect a possible unspecific cleavage either by autolysis or by proteolytic contaminations of the fusion protein.
|enzyme vol.(μl)||0, 0.5, 1, 2|
|100μg control protein||X|
|10X Cleavage buffer||10|
2. Incubate the reaction mixture at 4℃ for 16 hours or overnight.
3. Take out 20μl sample and add 20μl 2XSDS-PAGE loading buffer for each treatment and store at -20℃ until SDS-PAGE analysis. If practical, take out aliquots at different time spots to optimize the incubation time.
4. Determine and compare the extent of cleavage of the samples by SDS-PAGE analysis.
If shorter incubation time is required, more amount of HRV 3C protease or higher temperature (RT) can be implemented.
When the cleavage conditions are optimized at a small scale, scale up the
cleavage proportionally according to specific application requirement.
If IMAC Ni-charged resin is used after cleavage to remove the HRV 3C protease, the buffer of target protein should be exchanged into suitable buffers without EDTA or imidazole. Buffer exchange can be carried out by desalting or dialysis.
Fig. The control protein was cleaved by HRV 3C protease at 4°C for 16 h.
|Egg White cystatin||8μm||100|
 Cordingley, M. G., Register, R. B., Callahan, P. L., Garsky, V.M., and Colonno, R. J. (1989) Cleavage of Small Peptides In Vitro by Human Rhinovirus 14 3C Protease Expressed in Escherichia coli. J. Virol. 63, 5037-5045
 Q.May Wang and Shu-Hui Chen. (2007) Human Rhinovirus 3C Protease as a Potential Target for the Development of Antiviral Agents. Current Protein and Peptide Science, 8: 18-27
 Matthews, D.A., Smith, W.A., Ferre, R.A., Codon, B., Budahazi,G., Sisson, W., Villafranca, J.E., Janson, C.A., McElroy, H.E.,Gribskov, C.L. and Worland S. (1994) Structure of Human Rhinovirus 3C Protease Reveals a Trypsin-like Polypeptide Fold, RNA-Binding Site, and Means for Cleaving Precursor Polyprotein. Cell, 77, 761-771.
 Q. May Wang, Robert B. Johnson. (2001) Activation of Human Rhinovirus-14 3C Protease. Virology 280, 80-86