The endoprotease Factor Xa Protease removes vector-encoded amino acids from proteins expressed using the pQE-30 Xa vector (see page 74). Xa Removal Resin removes Factor Xa Protease from protein digests in a batch procedure.
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 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 "Digestion of 6xHis-Tagged Thioredoxin with Factor Xa Protease").
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 6xHis-tagged protein can be captured by Ni-NTA affinity chromatography.
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:
Digestion of 6xHis-Tagged Thioredoxin with Factor Xa Protease
|
Each reaction contained 10 µg of Ni/NTA-purified 6xHis-tagged thioredoxin expressed from pQE-30 Xa vector in E. coli and the indicated amount of Factor Xa Protease. Reactions were incubated at room temperature for 16 hours and analyzed on a Coomassie Blue-stained 15% polyacrylamide- SDS gel. M: markers. |