Fusion tag removal from the recombinant peptide fragment or protein that is preceded by the characteristic sequence DDDDK

 

Cleavage or processing of engineered proteins that contain enzyme recognition sequence.

 

Processing enzymes and proteins that are unstable in room temperature

 

Enterokinase will not work if the recognition site is followed by a proline.

 

rbovine EK with 6 × His-tag binds with Ni2+ affinity resin and was designed for removing from digestion system.

PreScission Protease is a fusion protein of human rhinovirus (HRV) 3C protease and GST. HRV 3C Protease is encoded by human rhinovirus 14.
It allows for on-column cleavage of GST tags and protein purification in one step.
 

 

Specific cleavage – between the Gln and Gly residues of the recognition sequence LeuGluValLeuPheGln/GlyPro.

• Highly active cleavage

• No non-specific proteolysis (highly specific cleavage)

• Activity from 2°C to 37°C

• A six-histidine sequence to facilitate its removal from the digested protein sample

1. Configurate the following reaction system in the EP tube:

Fusion protein:1mg

TEV Protease:10U
 

2. Incubate at suitable temperature after mixing the above reaction system. It is recommended that the reaction conditions of enzyme digestion is recommended at 4℃ overnight.
 

3. After the enzyme digestion, a small amount of samples can be selected for SDS-PAGE analysis.If you need to remove the TEV Protease in the reaction system after the enzyme digestion,using histidine tags purification resin affinity chromatography .

TEV Protease contains a 6x His-Tag for easy removal from a reaction using Ni agarose Resin (engibody,Cat no: P3403), or Ni-NTA Magnetic Beads (engibody,Cat no: P0030)
 

• Dextrin Beads 6FF is a robust, high resolution medium based on the 90 μm agarose matrix
 

• The small, evenly sized beads ensure that MBP-tagged Proteins elute in narrow peaks, thus minimizing the need for further concentration steps
 

• Physiological conditions and mild elution preserve target Protein activity
 

• Dextrin Beads 6FF tolerates all commonly used aqueous buffers and is easily regenerated using 0.5 M NaOH allowing the same column to be used for repeated purifications

• Stable coupling of ligand, low leakage; can be re-used to purify the same protein

L-Glutathione (GSH) reduced has been used in the elution buffer to elute GST (glutathione S-transferase)-fused proteins using glutathione-agarose beads. It has been used to prepare a standard curve for GSH analyses.

 

May be used at 5-10 mM to elute glutathione S-transferase (GST) from glutathione agarose.

His-tagged Protein Purification ( by use Ni-NTA Magnetic Beads)