Factor Xa Protease

If necessary, concentrate the fusion protein to at least 1 mg/ml.

Do a pilot experiment with a small portion of your protein.

Example: Mix 20 µl fusion protein at 1 mg/ml, with 1 µl Factor Xa diluted to 200 µg/ml, or 0.2 ng Enterokinase

In a separate tube, place 5 µl fusion protein with no protease (mock digestion). Incubate the tubes at room temperature. At 2, 4, 8, and 24 hours, take 5 µl of the reaction, add 5 µl 2x SDS-PAGE Sample Buffer, and save at 4°C. Prepare a sample of 5 µl fusion protein + 5 µl 2X sample buffer (uncut fusion).

Boil the 6 samples for 5 minutes and run on an SDS-PAGE gel.

Scale the pilot experiment up for the portion of the fusion protein to be cleaved. Save at least a small sample of the uncut fusion as a reference.

Check for complete cleavage by SDS-PAGE.

Denaturing the Fusion Protein

1. Either dialyze the fusion against at least 10 volumes 20 mM Tris-HCl, 6 M guanidine hydrochloride, pH 7.4 for 4 hours, or add guanidine hydrochloride directly to the sample to give a final concentration of 6 M.

2. Dialyze against 100 volumes Column Buffer, 2 times at 4 hours each.

During refolding, one has to balance between two objectives. For the protease to cleave it must be present before the protein has completely refolded, so removing the denaturant quickly is desirable. However, when the denaturant is removed quickly some proteins will fail to refold properly and precipitate. Stepwise dialysis against buffer containing decreasing amounts of guanidine hydrochloride can prevent precipitation of the fusion protein; halving the guanidine concentration at each step is convenient, but cases where 0.1 M steps are necessary have been reported. However, if the fusion protein is able to refold into a protease-resistant conformation, it may be better to dialyze away the denaturant in one step and take the loss from precipitation in order to maximize the amount of cleavable fusion protein recovered.

Go to step 2 or 4 above, as appropriate.