The expression of recombinant proteins as translational fusions is commonly employed to enhance stability, increase solubility and facilitate purification of the desired protein. There are several different kinds of fusion tags, such as GST, His, Protein A, MBP, TAP, E-tag, biotin, etc., but they all need to be removed after purification because the tag may affect biological function. Removing tags is an inherent problem with all fusion protein systems.
It is also a problem that I have had in my work. Recently, I successfully cloned, expressed and purified a protein in the pET42a vector as a GST fusion protein. A Factor Xa recognizes site was designed between the tag and my protein. Factor Xa protease is a site-specific endoprotease that preferentially cleaves the C-terminal peptide bond of the recognition sequence Ile-Glu-Gly-Arg. It consists of two polypeptides linked by a disulfide bond. On an SDS-PAGE gel, the reduced chains have an apparent molecular weights of 30 kDa and 20 kDa.
After searching around for the right kit, I decided to try the Factor Xa Cleavage Capture Kit from Novagen. It was not only the least expensive kit I had found, but it also included a convenient, affinity-bead based method for capturing and removing the active protease. In these types of experiments, one of the most important elements is ensuring that there the cleavage is extremely specific. To achieve this, it’s important that the protease is highly purified and there are no traces of contaminating proteases. The Factor Xa protease that comes with the Novagen kit is purified from bovine plasma and needs to be activated by Russell’s viper venom. In experiments where I’ve run the protease out on an SDS-PAGE gel, I have never found contaminating bands in Novagen’s Factor Xa. One issue to keep in mind is the need for optimizing cleavage conditions. In my initial experiments, I used too much protease which resulted in my protein being degraded. The conditions that need to be optimized include: Factor Xa Protease concentration, temperature (4° to 37°C), and incubation time (2 to 16 h). Based on the protocol posted on the Novagen website, I optimized my experiments on a small scale first then scaled up. Doing this, I got my experiments to work and purified my protein of interest.
Overall, this kit is very easy to use. This is especially true of the capture and removal beads (for removing the Factor Xa protease), which work in the same buffer as the protein cleavage reaction. The beads are washed once with the 1 cleavage/capture buffer, added to the cleavage reaction and incubated for about 10 minutes at room temperature. After the incubation, the beads are spun down and 99% of Factor Xa has been removed. Once again, the hardest part of the process is optimizing the conditions. This is because Factor Xa is sensitive to PBS, imidazole, NaCl (at concentrations of more than 250 mM) and SDS (which will cause secondary cleavage). Novagen suggests that the concentration of the target protein be at least 2.5uM in a reaction volume of 80ul or less. In my experience, it is very important to dialysis our protein in cleavage buffer and concentrate the protein before the cleavage reaction. Our target protein works better in pH 7.5 Tris and for optimal activity the proper temperature is important. Fortunately, Factor Xa is active at 4oC, so occassionaly, I let the cleavage reaction proceed in the cold room overnight (or longer).
The Factor Xa kit comes with 400 units of Factor Xa, (which is enough to treat 20 mg of recombinant protein), 2 ml Xa dilution and storage buffer (for small scale optimization experiments), 5 ml Xarrest Agarose (100ul is enough for removing 4 units of Xa), 10 spin filters with a 2ml capacity (surprisingly, the column is not enough. I solved this problem by spinning 30 seconds and pipetting up the supernatant carefully, without column is OK), 5 ml 10 cleavage/capture buffer (also not enough, but it is simple to make ourselves, 500mM Tris pH8.0, 1ml NaCl, 50mM CaCl2).
Overall, the Factor Xa Cleavage Capture Kit from Novagen is simple, easy to manipulate and cost efficient.
Xiang Yang, Ph.D.
Post-Doctoral Fellow
Georgetown University