Thermo Scientific Pierce Factor Xa is a serine endopeptidase composed of two disulfide-linked subunits, whose specificity makes it particular useful in cleaving 6xHis tags from engineered His-tagged fusion proteins.
Factor Xa is a serine endopeptidase composed of two disulfide-linked subunits that converts prothrombin to thrombin in the blood coagulation cascade. Factor Xa cleaves after the arginine residue in its preferred cleavage site Ile-(Glu or Asp)-Gly-Arg and it will occasionally cleave at other basic residues. However, it will not cleave at a site followed by proline or arginine.
- Remove histidine tags from fusion proteins
- Cleaves after the last amino acid in the sequence Ile-Glu-Gly-Arg
- Activity is > 125 U/mg using a synthetic Ile-Glu-Gly-Arg peptide with p-nitroanilide derivatized arginine as the substrate; each unit of enzyme cleaves 1 µmole/minute of substrate at 37°C in
5 mM MES, 0.5 M NaCl, 1 mM benzamidine at pH 6 with 1% BSA
Factor Xa is often used to remove fusion tags, such as the common histidine tag, from expressed proteins. By treating a purified, 6xHis-tagged protein expressed with a factor Xa cleavage site, it is possible to obtain the protein in its native form. The purified, expressed protein is incubated with factor Xa protease. After protease digestion, the protein of interest is repurified in two steps: removal of factor Xa protease followed by capture of histidine-tagged peptides and undigested histidine-tagged protein with an IMAC resin.
Although it is not always necessary to remove 6xHis tags from recombinant proteins, there are some applications, such as structural analysis by X-ray crystallography or NMR, where tag removal may be required. It may also be desirable to remove the histidine tag in other instances where the presence of this tag raises questions regarding the folding and function of the tagged protein.
|Properties of Factor Xa Protease.
||Coagulation factor Xa, prothrombase, prothrombinase, thrombokinase
||Cleaves primarily after the last amino acid in the sequence Ile-Glu-Gly-Arg
|Factor Xa Source
||Used as a cleavage reagent to remove GST tags from recombinant proteins
||pH 6-7, 37°C
||Aprotinin, PMSF, DFP, leupeptin
- Jesty, J. and Nemerson , Y. (1976) The activation of bovine coagulation factor X. Methods Enzymol. 45, 95-107.
- Nagai, K. and Thogersen, H. (1984) Nature 309, 810-812.
- Aurell, L., et al. (1984) Thrombosis Res.11, 595-609.
All proteases and protein-cleaving reagents (including mass spectrometry-grade)
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