Use of the Glu-Glu-Phe C-terminal epitope for rapid purification of the catalytic domain of normal and mutant ras GTPase-activating proteins.
The C-terminal catalytic domain (residues 704-1047) of the human ras GTPase-activating protein (GAP) has been engineered so as to incorporate the tripeptide, Glu-Glu-Phe, at its C terminus. This motif is recognized by the commercially available YL1/2 monoclonal antibody to alpha-tubulin and has previously been used for the immunoaffinity purification of HIV enzymes engineered to contain this epitope (Stammers, D. K., Tisdale, M., Court, S., Parmar, V., Bradley, C., and Ross, C. K. (1991) FEBS Lett. 283, 298-302). The engineered GAP catalytic domain (GAP-344) was obtained in high yield and purity from Escherichia coli extracts by means of a single affinity column of immobilized YL1/2, eluted under mild conditions with the dipeptide, Asp-Phe. The protein had similar activity to that previously described for full-length GAP, suggesting that the addition of the epitope did not grossly affect the activity. R903K and L902I mutants of GAP-344 were constructed, and the immunoaffinity purification procedure allowed their rapid characterization. The R903K mutant had less than 3% the activity of the normal protein, whereas the L902I substitution had less than 0.5% of normal activity, suggesting an important role for Leu-902 and Arg-903, residues absolutely conserved among GAP-related proteins. This work exemplifies the general utility of the C-terminal Glu-Glu-Phe motif for the rapid purification of proteins whose function is not altered by C-terminal modification.
Skinner
R H
RH
Department of Cell Biology, Wellcome Research Laboratories, Beckenham, Kent, United Kingdom.
Bradley
S
S
Brown
A L
AL
Johnson
N J
NJ
Rhodes
S
S
Stammers
D K
DK
Lowe
P N
PN
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics; D020690:GTPase-Activating Proteins; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification; D020703:ras GTPase-Activating Proteins
D000939:Epitopes; D020690:GTPase-Activating Proteins; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D004247:DNA