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Xa Removal Resin

For removal of Factor Xa Protease after protease digestion
  • Specific and efficient removal of Xa protease
  • Results in high purity of the target proteins

After purification of a 6xHis-tagged protein expressed using pQE-30 Xa vector and cleavage of the 6xHis tag using Factor Xa Protease, the protein of interest can be repurified in two steps. Xa Removal Resin binds Factor Xa Protease in a batch procedure, and is removed by centrifugation. Cleaved 6xHis-tag peptides and undigested 6xHis-tagged protein are captured by Ni-NTA affinity chromatography, the purified protein of interest being in the flow-through.

The Xa Removal Resin is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
Performance
Xa Protease Resin specifically removes Xa Protease leaving cleaved target protein and any uncleaved His-tagged protein in the supernatant after centrifugation (see figure "Digestion of 6xHis-tagged thioredoxin").
Principle

Although it is rarely necessary to remove the short 6xHis affinity tag from a recombinant protein after purification, there are some applications, such as structural analysis by X-ray crystallography or NMR, where removal of the tag may be desirable.

The expression vector pQE-30 Xa encodes a Factor Xa Protease recognition site between the N-terminal 6xHis-tag sequence and the multiple cloning site. Factor Xa Protease recognizes the amino acid sequence Ile-Glu-Gly-Arg and cleaves the peptide bond C-terminal of the arginine residue. If the gene of interest is cloned blunt-ended at the 5' end using the Stu I restriction site of the vector, Factor Xa Protease cleavage of the purified recombinant protein results in a protein product without any vector-derived amino acids at the N-terminus (see figure "Digestion of 6xHis-tagged thioredoxin"). The Factor Xa Protease can be removed by being bound by Xa Removal Resin in a batch procedure followed by centrifigation.

Procedure

The purified, 6xHis-tagged, expressed protein is incubated with Factor Xa Protease. After 6xHis-tag cleavage, the protein of interest is repurified in two steps. Xa Removal Resin binds Factor Xa Protease in a batch procedure, and is removed by centrifugation. Subsequently, cleaved 6xHis-tag peptides and undigested 6xHis-tagged protein can be captured by Ni-NTA affinity chromatography. The purified protein of interest appears in the flow-through fraction.

Applications

Proteins processed using the Factor Xa system are well suited for applications where the removal of a protein’s His tag may be preferred, including:

  • Protein crystallography
  • Structure determination studies using NMR
Features
Specifications
Applications Proteomics
Binding capacity 50 µl bed volume/4U enzyme
Gravity flow or spin column Spin column
Processing Manual
Start material Protein
Support/matrix Xa removal matrix
fragment fix placeholder