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HRV-3C Protease Cleavage Enzyme, GST Tag  pdf  pdf  pdf


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3CC-N3136-1000U
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Kit Content

1KU or 2.5KU or 50KU Recombinant HRV 3C Protease ( lyophilized from 50mM Tris, 150mM NaCl, 1mM EDTA, 0.05% Tween20)
100 μg Cleavage Control Protein ( lyophilized from sterile PBS , pH 7.4 )
10 ml or 100ml 10X HRV 3C Cleavage Buffer (500mM Tris-HCl, pH-7.0, 1.5 M NaCl, 10 mM EDTA, 10 mM dithiothreitol )

Synonym

HRV 3C Protease, 3C, HRV-3C

Source

Recombinant GST-HRV 3C Protease is a recombinant form of human rhinovirus (HRV) type 14 3C protease (22KDa on SDS-PAGE) produced in Escherichia coli cells at ACRObiosystems.

Application

The high specificity of GST-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 GST-HRV 3C to obtain the target protein.

Unit Definition

>1 Units/µg. One unit will cleave >95% of 100 µg control fusion protein in 50 mM Tris-HCl, 150 mM NaCl, pH 7.5 at 4oC for 16 h.

Specificity

The enzyme recognizes the cleavage site: Leu-Glu-Val-Leu-Phe-Gln-↓-Gly-Pro.

Formulation

Lyophilized from 0.22 μm filtered solution in 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.05% Tween20. Contact us for customized product format or formulation.

Reconstitution

See Certificate of Analysis for details of reconstitution instruction and specific concentration.

10×Cleavage Buffer

500mM Tris-HCl, pH-7.0, 1.5 M NaCl, 10 mM EDTA, 10 mM dithiothreitol. Chill to 5oC prior to use.

Storage

Store at -20oC after reconstituted. Avoid repeated freeze-thaw cycles.

Example

HRV-3C Activity Example

Cleavage Protocol

  • Make fresh cold cleavage buffer ,recommended typical cleavage buffer is : 50 mM Tris-HCl, pH-7.0 (at 25℃), 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol. Cleavage buffer should be a buffer in which the target protein is soluble. There should be no protease inhibitor in the buffer. The cleavage 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. HRV-3C Protease is compatible with  500 mM NaCl and 400 mM imidazole.
  • Dilute the fusion protein pool to 1-2 mg/ml with cold Cleavage Buffer. This is optional in case the target protein aggregates in the buffer. Keep a small aliquot as Uncut sample(Negative Control) to detect a possible unspecific cleavage either by autolysis or by proteolytic contaminations of the fusion protein.
  • Add GST-HRV 3C protease at a Protease : Target protein ratio of 1 :100 (w/w) (1,000 unit GST-HRV 3C Protease to 100 mg target protein) as initial cleavage condition. The optimal ratio should be determined empirically. A Protease-to-target protein ratio (w/w) of 1:50 to 1:400 should work for most target proteins. There is no need to change buffer or dilute HRV-3C Protease.
  • Use150mM NaCl sterile solution to dilute DNA plasmid and add 293 expression MAX-1 according separate manual add BPfectin at ration of 3uL:1ug(BPfectin:DNA) drop wise to DNA solution and vertex for 3 min; drop-DNA dosage for transfection is 1ug per 1 million cells in culture, and volume of DNA-BPfectin complexes is 5% of culture to be transfected.
  • Incubate the reaction mixture at 4℃ for 16 hours or overnight. If shorter incubation time is required, more amount of HRV-3C protease or higher temperature (RT) should be implemented. When the cleavage conditions are optimized at a small scale, scale up the cleavage proportionally according to specific application requirement.

Removal of HRV GST-3C Protease:

  • Use Glutathione Agarose High Flow (ACROBiosystems Catalog# GS-0207-01) to remove the HRV GST-3C Protease
  • If desired, determine and compare the extent of cleavage of the samples by SDS-PAGE analysis.

Experimental Outline:

3CC-N3136-Flow

Activity Influence Factors:

Depending on the buffers used and their chemical components, HRV GST-3C Protease cleavage efficiency may be affected. The following table shows the relative activity of HRV GST-3C Protease under various conditions.

3CC-N3136-Factor


Background

Protease Recombinant is fusion protein of GST and human rhinovirus (HRV) type 14 3C protease.Substrate recognition and cleavage are likely to be dependent not only upon primary structural signals, but also upon the secondary and tertiary structures of the fusion protein as 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.

Please contact us via TechSupport@acrobiosystems.comif you have any question on this product.

References

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