Factor Xa Protease
For endoproteolytic removal of short 6xHis affinity tags from 6xHis-tagged recombinant proteins
Factor Xa Protease is a site-specific endoprotease that preferentially cleaves the C-terminal peptide bond of the recognition sequence Ile-Glu-Gly-Arg. Therefore, Factor Xa Protease efficiently cleaves 6xHis-tagged proteins expressed by the pQE-30 Xa vector as the 6xHis tag precedes the recognition sequence, giving protein free of vector-encoded amino acids. After cleavage of the purified protein, Factor Xa Protease is removed in a batch procedure using Xa Removal Resin.
The Factor Xa Protease is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
Performance
Factor Xa Protease efficiency cleaves the 6xHis tag when expressed adjacent to the peptide sequence Ile-Glu-Gly-Arg (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").
Procedure
The purified, expressed protein with a 6xHis-tag is incubated with Factor Xa Protease. After protease digestion, 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 digested protein without the His-tag 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:
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