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 agarose. It was apparent from reverse phase chromatography that the purified protein was conformationally unstable. Instability was overcome by the addition of 100 mM arginine to the buffers. Because Grb2 appeared to be extremely sensitive to oxidation, crystallization experiments were performed with a dialysis button technique involving daily addition of fresh DTT to the reservoirs. The presence of 8 to 14% glycerol was necessary to obtain monocrystals. These results are discussed in relation with the modular nature of Grb2.
Guilloteau
J P
JP
Service de Biochimie, Rhône-Poulenc Rorer SA, Vitry/Seine, France.
Fromage
N
N
Ries-Kautt
M
M
Reboul
S
S
Bocquet
D
D
Dubois
H
H
Faucher
D
D
Colonna
C
C
Ducruix
A
A
Becquart
J
J
D048868:Adaptor Proteins, Signal Transducing; D001120:Arginine / Q000494:pharmacology; D015374:Biosensing Techniques; D002846:Chromatography, Affinity; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors; D004926:Escherichia coli / Q000235:genetics; D051380:GRB2 Adaptor Protein; D005982:Glutathione Transferase / Q000235:genetics; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D013438:Sulfhydryl Compounds / Q000032:analysis; D013917:Thrombin / Q000378:metabolism; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D001120:Arginine; D005982:Glutathione Transferase; D066246:ErbB Receptors; D013917:Thrombin