Minimal FLAG sequence useful in the functional epitope tagging of H-Ras.
Epitope tagging can interfere with normal protein function, indicating the need for an unobtrusive epitope tag. The FLAG epitope (DYKDDDDK) was examined for a minimal epitope useful in the tagging of H-Ras. The heptapeptide tag, F7 (MDYKDDD), was found to retain reactivity with M2 and M5 monoclonal antibodies in immunoprecipitation, Western blotting, and immunofluorescence microscopy. The F7 tag did not interfere with Ras stability, EGF stimulation of Ras activation, and downstream phosphorylation of MAPK Erk1/2. Unlike the full FLAG sequence, the F7 tag had minimal effect on the growth properties of H-Ras in a colony-forming assay. The F7 tag may be useful when minimizing the effect of tagging on protein function is an important criterion in the selection of an N-terminal epitope tag.
Johnson
Kristy Y
KY
Medical University of South Carolina, Charleston 29425, USA.
Liu
Liya
L
Vincent
Timothy S
TS
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D000939:Epitopes / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D011905:Genes, ras; D051379:Mice; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics*; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection; D018631:ras Proteins / Q000235:genetics / Q000276:immunology*
D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide; D018631:ras Proteins