TY - JOUR
T1 - Diverting a protein from its cellular location by intracellular antibodies
T2 - The case of p21Ras
AU - Lener, Matteo
AU - Horn, Ivo R.
AU - Cardinale, Alessio
AU - Messina, Samantha
AU - Nielsen, U. B.
AU - Rybak, Susanne M.
AU - Hoogenboom, Hennie R.
AU - Cattaneo, Antonino
AU - Biocca, Silvia
PY - 2000
Y1 - 2000
N2 - We describe the use of phage libraries to derive new antibodies against p21Ras to be used for intracellular expression in mammalian cells. A panel of single-chain antibody fragments, binding to Ras, were analyzed and characterized for their capacity to interfere in vitro with (a) the intrinsic GTPase activity of Ras and (b) the binding of Ras to its effector Raf, and were found not to neutralize its function, according to these biochemical criteria. When expressed intracellularly in mouse 3T3 K-Ras transformed cells all the anti-Ras single-chain variable fragments (scFv) tested inhibited cell proliferation, as assessed by bromodeoxyuridine incorporation. Double immunofluorescence analysis of transfected cells using confocal microscopy confirmed that anti-Ras antibody fragments colocalize with endogenous Ras, at subcellular locations where the protein Ras is not normally found. These data suggest that the ability of phage-derived anti-Ras scFv fragments to inhibit the function of Ras in vivo is a rather general and frequent property and that the range of antibodies that can be successfully used for intracellular inhibition studies is much greater than anticipated, exploiting the mode of action of diverting protein traffic.
AB - We describe the use of phage libraries to derive new antibodies against p21Ras to be used for intracellular expression in mammalian cells. A panel of single-chain antibody fragments, binding to Ras, were analyzed and characterized for their capacity to interfere in vitro with (a) the intrinsic GTPase activity of Ras and (b) the binding of Ras to its effector Raf, and were found not to neutralize its function, according to these biochemical criteria. When expressed intracellularly in mouse 3T3 K-Ras transformed cells all the anti-Ras single-chain variable fragments (scFv) tested inhibited cell proliferation, as assessed by bromodeoxyuridine incorporation. Double immunofluorescence analysis of transfected cells using confocal microscopy confirmed that anti-Ras antibody fragments colocalize with endogenous Ras, at subcellular locations where the protein Ras is not normally found. These data suggest that the ability of phage-derived anti-Ras scFv fragments to inhibit the function of Ras in vivo is a rather general and frequent property and that the range of antibodies that can be successfully used for intracellular inhibition studies is much greater than anticipated, exploiting the mode of action of diverting protein traffic.
KW - Anti-p21Ras
KW - Intracellular antibodies
KW - Phage display library
KW - scFv fragments
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U2 - 10.1046/j.1432-1327.2000.01125.x
DO - 10.1046/j.1432-1327.2000.01125.x
M3 - Article
C2 - 10672031
AN - SCOPUS:0033952543
VL - 267
SP - 1196
EP - 1205
JO - European Journal of Biochemistry
JF - European Journal of Biochemistry
SN - 0014-2956
IS - 4
ER -