Diverting a protein from its cellular location by intracellular antibodies. The case of p21Ras.
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.
Lener
M
M
Department of Neuroscience, University of Rome 'Tor Vergata', Roma, Italy.
Horn
I R
IR
Cardinale
A
A
Messina
S
S
Nielsen
U B
UB
Rybak
S M
SM
Hoogenboom
H R
HR
Cattaneo
A
A
Biocca
S
S
D016475:3T3 Cells; D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D000915:Antibody Affinity; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D001692:Biological Transport / Q000187:drug effects; D019556:COS Cells; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D005455:Fluorescent Antibody Technique; D006868:Hydrolysis / Q000187:drug effects; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D009500:Neutralization Tests; D019151:Peptide Library; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism*; D014162:Transfection
D000906:Antibodies; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D004247:DNA; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras)