pQE-30 Xa Vector
For expression of His-tagged proteins containing a Factor Xa Protease recognition site
The pQE-30 Xa vector is similar to pQE-30, but also encodes a Factor Xa Protease recognition site, which is bracketed by the His tag coding region on the 5' side and the multiple cloning site on the 3' side. 5'-end cloning using the blunt-end StuI restriction site allows insertion of the gene of interest directly behind the Factor Xa Protease recognition site, without any intervening amino acid codons. Factor Xa Protease cleaves off the His tag peptide behind the arginine residue of the protease recognition site (IEGR) and results in a recombinant protein free of any vector-derived amino acids at the N-terminus. After digestion, Factor Xa Protease can be removed using Xa Removal Resin.
The pQE-30 Xa vector was used to express His-tagged thioredoxin, whose tag was then cleanly removed by Factor X protease (see figure Digestion of His-tagged thioredoxin with Factor Xa protease).
Although it is rarely necessary to remove the short His 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 His 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 StuI 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 pQE-30 Xa).
The purified, expressed protein 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 His-tagged protein can be captured by Ni-NTA affinity chromatography.
Proteins processed using the Factor Xa System, which uses the pQE-30 Xa vector, are well suited for applications where the removal of a protein’s His tag may be preferred, including:
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