Thrombin cleaved GST-fusion protein degredation--suggestions - (Nov/12/2008 )
I am trying to purify my protein of interest from a GST-fusion using thrombin to cleave my protein from the gultathione beads. The resulting elution contains a strong band at the correct size (90 kDa) followed by many smaller bands which I am assuming are degradation products. I am using PMSF, DTT, and the 25X complete cocktail to inhibit protease activity but with seemingly little success. The lysis is done using a French press. I am hoping to do x-ray crystallography with my protein and need a very pure sample to do so if anyone has any suggestions I would be very appreciative.
You may want to try to do all the purification at 4oC to reduce degradation. What are the sizes of the smaller bands? Very close to your protein? If not, maybe can get rid of them by dialyse and/or filter. You need to get rid of the thrombin anyway.
Time course at 2 or 3 temperatures. Collect fractions every 10-15 minutes at 10C, RT and 37.