Hi Xtallographers, I have been able to purify a protein that was initially going into inclusion bodies from the excellent suggestions that I got here.
So my lysis buffer has 0.5M Guanidium Hydrochoride, 2% TritonX-100, 500mM NaCl, 5% Glycerol in 20 mM Tris-HCL pH 8.0 The problem is that the protein is first purified as a SUMO-fusion protein which is further proteolysed and passed through the Talon resin to get the final SUMO-Free construct. However as I have around 250mM Imidazole (pH elution did not work) from the elution of the first round, I have to dialyse the sample to get rid of the imidazole so that I can use the proteolysed sample again on the column. All these I do in a buffer that does not have GuHCL or Triton. However I have kept the NaCl concentration same (0.5 M). I start to see white insoluble precipitate right from the dialysis step. If I spin the precipitate out, I still have a lot of protein to go to the next step of proteolysis. The problem is that when I finally want to concentrate the protein to run the SEC step, all my protein starts precipitating starting from 5mg/ml to all the way to 25-30 mg/ml. Are there some smart ways to keep the protein soluble at higher concentrations, assuming that I do not have to remove them before setting up trays? Should I keep on using Guanidium Hcl and Triton for all the steps all the way into the SEC column. Have people got any good results using 5% Acetronitrile, 50mM Arginine or DTT? (used for NMR samples) Any help in this regard will be very helpful. The protein is an engineered bacterial transcription factor. (not a membrane protein) Thanks in advance as always, ivan
