|1KU or 2.5KU||HRV-3C Protease Cleavage Enzyme, GST Tag, His Tag ( lyophilized from 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.05% Tween20)|
|100 μg||Cleavage Control Protein ( lyophilized from sterile PBS , pH 7.4 )|
|25ml||10X HRV 3C Cleavage Buffer (500 mM Tris-HCl, pH7.0, 1.5 M NaCl, 10 mM EDTA, 10 mM dithiothreitol )|
The enzyme recognizes the cleavage site: Leu-Glu-Val-Leu-Phe-Gln-↓-Gly-Pro.
Activity Influence Factors
Make fresh cold cleavage buffer ,recommended typical cleavage buffer is : 50 mM Tris-HCl, pH7.0 (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.
Use Glutathione Agarose High Flow to remove the HRV GST-3C Protease
If desired, determine and compare the extent of cleavage of the samples by SDS-PAGE analysis.
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.
Authors:Jakubczyk D, et al.
Journal:Chem Commun (Camb) 2016
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