Characterization of the Ras-related RAP2A protein expressed in the baculovirus-insect cell system: processing of the protein in insect cells and comparison with the bacterially produced unprocessed form.
The Ras-related protein Rap2A is a 21kD GTP-binding protein that exhibits 46% identity with Ras proteins and is similarly post-translationally modified by farnesyl and palmitate groups. Using a recombinant baculovirus, we expressed Rap2A in Sf9 insect cells. The protein is initially synthesized as a soluble hydrophilic precursor, that is post-translationally processed to a hydrophobic membrane-bound form (Rap2Am) that contains both isoprenoid and palmitate groups. The processed form of the protein was purified from the membranes of infected Sf9 cells, and its biochemical properties were compared with those of the unprocessed form produced in recombinant bacteria (Rap2Ab). Both proteins exhibited similar kinetics of GDP dissociation and GTP binding and displayed a weak intrinsic GTPase activity that was stimulated to the same extent by a factor present in bovine brain cytosol. We conclude that Rap2A is correctly processed in insect cells and that maturation does not alter its biochemical properties.
Pasheva
E
E
INSERM U-248, Faculté de Médecine Lariboisière-Saint Louis, Paris, France.
Janoueix-Lerosey
I
I
Tavitian
A
A
de Gunzburg
J
J
D000818:Animals; D016367:Baculoviridae; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D009036:Moths; D011499:Protein Processing, Post-Translational; D014162:Transfection; D020668:rap GTP-Binding Proteins
D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D008715:Methionine; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins