PURIFICATION, STABILIZATION, AND CRYSTALLIZATION OF A MODULAR PROTEIN- GRB2

We report here the purification and the crystallization of the modular protein Grb2. The protein was expressed as a fusion with glutathione- S-transferase and purified by affinity chromatography on glutathione a garose. It was apparent from reverse phase chromatography that the pur ified protein was conformationally unstable. Instability was overcome by the addition of 100 mM arginine to the buffers. Because Grb2 appear ed to be extremely sensitive to oxidation, crystallization experiments were performed with a dialysis button technique involving daily addit ion of fresh DTT to the reservoirs. The presence of 8 to 14% glycerol was necessary to obtain monocrystals. These results are discussed in r elation with the modular nature of Grb2. (C) 1996 Wiley-Liss, Inc.