Human rhinovirus 3C protease (HRV3C Protease) is a cysteine protease that recognizes the cleavage site of Leu-Glu-Val-Leu-Phe-Gln-h-1192_HRV3C cleavageGly-Pro. It cleaves between Gln and Gly. The recombinant form of the HRV3C Protease is a restriction grade protease that has robust activity at 4oC with high specific activity and great stability.

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. It does not require any special buffer for its activity and can be used in a buffer most suitable for target proteins.  This 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.

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 µg of target protein in a buffer suitable for the target protein at 4oC 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 HRV3C Protease 50-400 µg 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 specificity, more HRV3C Protease (at 1:10 ratio) or longer cleavage time (over a weekend) at higher temperature (37°C) can be used to achieve high cleavage efficiency without non-specific cleavage of target proteins.

The 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.

 

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

Turbo3C 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 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.

4. 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.

Removal of Turbo3C Protease

1. 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.

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.

 

Frequently asked Questions ( FAQS)

BACKGROUND ISSUE: I have had numerous problems with the expression of my protein, the foremost being copurification of a GST binding protein along with my protein of interest. This is particularly difficult as I am unable to sufficiently quantify my protein of interest due to the presence of this contaminating protein. I have included a HRV3c protease cleavage site between my GST and my protein of interest.

SO I am using Ni+ magnetic beads for my purification. I want to try 'on column cleavage to eliminate the use of glutathione for elution as this affects my downstream experiment using the protein. I had some questions about your product:

  • QUESTION: Can you tell me if the protease has been tested for 'on column cleavage' and what is its efficiency?

       ANSWER: Turbo3C Protease works with on column cleavage with less efficiency than in solution cleavage. More Turbo3C is needed. Start with injecting 0.2 mg/ml (200 units/ml) Turbo3C solution to the column for an overnight digestion at 4C. Adjust the enzyme concentration based on the results.

 

  • QUESTION: The magnetic bead purification protocol calls for buffer composed of 125 mM Tris pH8.0 and 150mM NaCl. I see the HRV protocol calls for 25-50 mM Tris. Has higher Tris concentrations been tested and does it affect the cleavage efficiency.

       ANSWER: The buffer is fine. Turbo3C Protease works with most buffers that a soluble protein is compatible.

 

  • QUESTION: Are there any compounds that inhibit cleavage?

       ANSWER: Turbo3C is a Cys protease, any compound that inhibits Cys protease will inhibit Turbo3C. So after binding, wash away any protease inhibitors introduced in the lysis buffer.

 

  • QUESTION: What reaction volume do you recommend for cleavage using 1 unit of HRV?

       ANSWER: On column cleavage requires high concentration of Turbo3C. We recommend 200 units/ml (0.2 mg/ml).

 

  • QUESTION: I realize different proteins will behave differently but due to the limited amount of protein that I have, I want to use the most ideal conditions for the optimization experiment. Any information (particularly any experience with cleaving insect cell expressed protein, if there is any difference) regarding cleavage conditions etc would be very helpful.

       ANSWER: In general, GST binding proteins from insect cells are not a problem for purification. But often GST-tag is more abundant than the GST-fusion. The big band around 23-30 kD on the gel is the GST-tag without the target protein. The solutions are changing the junction sequence or codon optimization of the target protein.

If the contaminating proteins have different sizes from the target protein, a size exclusion column (SEC) may solve the problem.

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