>HRV 3C Protease (Human Rhinovirus 3C Protease, PreScission Site)
|Catalog||Size (Price)||Quantity||In Stock||Operation||Other Information|
The recombinant human rhinovirus (HRV 3C) protease is used to remove fusion tags from proteins with the HRV 3C cleavage sequence (PreScission Site): Leu-Glu-Val-Leu-Phe-Gln↓Gly-Pro or LEVLFQ↓GP.
The recombinant HRV 3C protease is a fusion protein of 22-kDa size, with a his tag. The enzyme retains high activity at 4°C, and can cleave the target protein in solution, in dialysis or on column.
- High specificity: recognizes sequence Leu-Glu-Val-Leu-Phe-Gln-↓-Gly-Pro and cleaves after the glutamine residue.
- Retain high activity at 4°C: cleavage at low temperature preventing instability of target protein at high temperature.
- Easy removal: His-tagged protease can be easily removed after cleavage using a variety of solid supports with immobilized metal chelates.
- Lyophilized form: increases the stability and shelf life during storage and shipping, and decreases the shipping cost.
- Flexible: HRV 3C protease can cleave the target protein in solution, in dialysis or on column.
- Cost effective (cost saving): >50% cost saving, lower price, double size.
|Recombinant HRV 3C Protease||2000 U||lyophilized from 50mM Tris, 150mM NaCl, 1mM EDTA, 1mM DTT, 0.04% Tween20, 8% trehalose, 8% mannitol|
|Cleavage Control Protein||100 μg||lyophilized from sterile PBS , pH 7.4|
|10X HRV 3C Cleavage Buffer||5 ml||1.5 M NaCl , 0.5 M Tris-HCl , pH 7.5|
Activity Definition: One unit of HRV 3C Protease is defined as the amount of enzyme that will cleave > 95% of 100 μg Cleavage Control Protein in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5 at 4°C in 17 h.
Storage: HRV 3C protease at –20°C; cleavage control protein and cleavage buffer at –20°C or 4°C.
HRV 3C protease digest condition:
|Incubation time:||16 hours or overnight|
|Enzyme amount:||For most applications, 1:100–1:25 enzyme-to-substrate ratio (unit/μg)|
Small scale optimization:
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.
- Combine 100μg fusion protein, 10μl 10×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.
Component Volume (μl) enzyme volume (μl) X 100μg control protein Y 10X Cleavage buffer 10 Sterile water 100-X-Y
- Incubate the reaction mixture at 4℃ for 16 hours or overnight.
- Take out 20μl sample and add 20μl 2×SDS-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.
- Determine and compare the extent of cleavage of the samples by SDS-PAGE analysis.
The protease: target protein ratio can reach 1:800 in our experiment, but we recommend excessive use of the high specificity protease(1:100~1:25) to insure enzymatic digest completely in different experiments.
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. Optimization of HRV 3C protease cleavage at 4°C for 16 h. A MBP-fusion protein at 1.17mg/ml was cleaved at various ratios of HRV 3C protease to the fusion protein
|Molecular Weight:||~22KDa on SDS-PAGE||SDS-PAGE:
|Purity:||98% by SDS-PAGE.|
|Quality Control:||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.|
Factors that influence HRV 3C activity
Depending on the buffers used and their chemical components, HRV 3C Protease cleavage efficiency may be affected. The following table shows the relative activity of HRV 3C Protease under various conditions.
|Factor||Reagent||Concentration||Relative Activity (%)|
|Egg White cystatin||8μm||100|
|Incomplete cleavage||Suboptimal HRV 3C Protease to fusion protein ratio||Confirm the amount of fusion protein in the digestion. Adjust the amount of HRV 3C Protease added to at least 10 U/mg fusion protein.|
|Insufficient incubation period||Increase reaction time.|
|HRV 3C Protease recognition site not present or has been altered during the course of cloning.||Verify presence of optimal HRV 3C Protease cleavage sequence.|
|HRV 3C Protease recognition site is not accessible.||Reversibly denature protein with non-ionic detergents, denaturants .|
|HRV 3C Protease inhibitors present||Dialyze the fusion protein against Cleavage Buffer before cleaving with HRV 3C Protease.|
|On-column cleavage less than optimal.||Increase HRV 3C Protease concentration.|
|Perform cleavage in solution or dialysis.|
|HRV 3C Protease contamination after purification with Ni-NTA His•Bind® resin||Ni-NTA His•Bind Resin was saturated with His•Tag fusion protein or fusion tag. Removal of HRV 3C Protease by the resin was incomplete.||Increase amount of Ni-NTA His•Bind Resin or decrease amount of extract loaded on Ni-NTA His•Bind Resin.|
|Pass sample over freshly prepared or regenerated Ni-NTA His•Bind Resin.|
|Multiple bands present on SDS-PAGE Gel following cleavage by HRV 3C Protease||Similar secondary recognition sequences in protein of interest.||Adjust reaction conditions to minimize exposure of secondary cleavage sites.|
|Proteolysis at secondary sites due to excess HRV 3C Protease||Reduce HRV 3C Protease concentration.|
|Proteolysis in bacterial host||Use protease-deficient strain (e.g., lon or ompT), such as E. coli BL21(DE3).|
HRV 3C protease background
The catalytic activities of 3C protease have been found to be essential for generation of mature viral proteins and functional viral enzymes and thus for viral replication in the human rhinoviruses (HRVs) which are members of the picornavirus family and are the major causative agents of common cold. HRVs contain a single strand, positive-sense RNA genome which encodes a single viral polyprotein. This polyprotein precursor is further processed by two viral proteases designated 2A and 3C protease. It is believed that the 2A protease makes the first cleavage at its own N-terminus to separate the structural capsid proteins from the nonstructural ones, while the 3C protease processes most of the remaining sites.
Structure of HRV 3C protease
Sequence comparison, structure and mutational analysis, and studies with classic protease inhibitors have all shown that HRV 3C protease contains an active site cysteine residue as the nucleophile. However, its overall structures are more like serine proteases rather than the typical cysteine proteases. For HRV 3C protease, the catalytic triad is composed of His-Glu-Cys and is positioned as seen in serine proteases. HRV 3C protease will fold into two anti-parallel six-stranded β-barrels and the site cleft is located at the junction of the two β-barrels domains.
Activity of HRV 3C protease
In infected cells, HRV14 3C is able to process the polyprotein precursor at the scissile bonds formed between Gln-Gly, Gln-Ala, and Glu-Gly. However, in vitro studies have shown that this enzyme has a robust activity to cleave at the PreScission site (Leu-Glu-Val-Leu-Phe-Gln-↓-Gly-Pro) with high specificity. And the enzyme requires neither metal nor cofactors for activity. It has been demonstrated that the enzyme exhibits highest activity around neutral pH at temperatures ranging from 22 to 37℃, even retaining robust activity at 4℃.
Recombinant HRV 3C protease
Recombinant HRV 3C protease encoded by human rhinovirus 14 is a highly purified recombinant protease with a His-tag or GST-tag produced in E. coil system which has the activity as same as the wild type. The protease is a ~20kDa single-chain protein containing approximately 182 amino acids cover the 1538-1717 region of HRV 14's Genome polyprotein (Accession #: P03303) with calculated isoelectric point 8.46.
- MG Cordingley, RB Register, PL Callahan. (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, SH Chen. (2007) Human Rhinovirus 3C Protease as a Potential Target for the Development of Antiviral Agents. Current Protein and Peptide Science, 8: 18-27
- DA Matthews, WW Smith, RA Ferre. (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 Wang, RB Johnson. Johnson. (2001) Activation of Human Rhinovirus-14 3C Protease. Virology 280, 80-86