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., Horn, I.r., Cardinale, A., Messina, S., Nielsen, U.b., Rybak, S.m., et al. (2000). Diverting a protein from its cellular location by intracellular antibodies: The case of p21Ras. EUROPEAN JOURNAL OF BIOCHEMISTRY, 267(4), 1196-1205 [10.1046/j.1432-1327.2000.01125.x].

Diverting a protein from its cellular location by intracellular antibodies: The case of p21Ras

BIOCCA, SILVIA
2000-01-01

Abstract

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.
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore BIO/12
English
Con Impact Factor ISI
intracellular antibodies; scFv fragments; anti-p21Ras; phage display library
Lener, M., Horn, I.r., Cardinale, A., Messina, S., Nielsen, U.b., Rybak, S.m., et al. (2000). Diverting a protein from its cellular location by intracellular antibodies: The case of p21Ras. EUROPEAN JOURNAL OF BIOCHEMISTRY, 267(4), 1196-1205 [10.1046/j.1432-1327.2000.01125.x].
Lener, M; Horn, Ir; Cardinale, A; Messina, S; Nielsen, Ub; Rybak, Sm; Hoogenboom, Hr; Cattaneo, A; Biocca, S
Articolo su rivista
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/54225
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 62
  • ???jsp.display-item.citation.isi??? 61
social impact