p55-hGRF, a short natural form of the Ras-GDP exchange factor high yield production and characterization.
p55-hGRF, a natural short form of the guanine-nucleotide-releasing factor for p21-Ras from human brain, was expressed at high level in Escherichia coli as well as an engineered truncated form, p39-hGRF. A T7 polymerase expression system was used, resulting in the formation of insoluble cytoplasmic protein aggregates. The recombinant products were resolubilized, renatured and purified to homogeneity. The exchange activity of the refolded hGRF samples on H-Ras was comparable with that published for the soluble catalytic domain of the mouse counterpart, CDC25 Mm. Both p55-hGRF and p39-hGRF form dimers. We established a procedure to prepare and purify the complex with Ras. The results of the characterization study are consistent with a stoichiometry of 1:1 and an equilibrium between dimeric and monomeric forms of the complex.
Meyer
P
P
Laboratoire dEnzymologie et Biochimie Structurales, UPR 9063, CNRS, Gif sur Yvette, France.
Janin
J
J
Baudet-Nessler
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D019281:Dimerization; D004926:Escherichia coli; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D020663:ras Guanine Nucleotide Exchange Factors
D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D011994:Recombinant Proteins; D020663:ras Guanine Nucleotide Exchange Factors; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)