Bio/Technology
12, 396 - 399 (1994)
doi:10.1038/nbt0494-396
Intracellular Immunization with Cytosolic Recombinant AntibodiesSilvia Biocca1, *, Paola Pierandrei-Amaldi2, Nadia Campioni2
& Antonino Cattaneo1, 3, *
1Institute of Neurobiology, Viale K. Marx 15, 00137 Roma, Italy.
2Institute of Cell Biology, CNR, Viale K. Marx 15, 00137 Roma, Italy.
3International School for Advanced Studies (SISSA), Trieste, Italy.
*Corresponding authors at: Institute of Neurobiology, Viale K. Marx 15, 00137 Roma, Italy. We report the application of a strategy to inactivate cellular proteins in vertebrate cells based on the intracellular expression of immunoglobulin genes. We have selected, in this instance, the p21 protein, encoded by the ras protooncogene, as a target protein. The variable regions of the neutralizing anti-p21ras monoclonal antibody Y13-259 were cloned in vectors for the expression of either the whole antibody molecule or its singlechain Fv fragment (ScFv) derivative. In order to target the recombinant antibodies to the cytosol, their hydrophobic leader sequence for secretion was mutated or deleted. When these proteins are expressed in the cytosol of Xenopus laevis oocytes they colocalize with the endogenous p21ras protein in the cytoplasmic face of the oocyte plasma membrane, and they markedly inhibit the H1 kinase activity induced by insulin. Moreover, cytosolic anti-p21ras ScFv fragments block the ensuing meiotic maturation. Thus the intracellular expression of both whole antibodies and antibody domains can be used to block a biological function.
REFERENCES
- Carlson, J.R. 1988. A new means of inducibly inactivating a cellular protein. Mol. Cell. Biol. 8: 2638−2646. | PubMed | ISI | ChemPort |
- Biocca, S., Neuberger, M.S. and Cattaneo, A. 1990. Expression and targeting of intracellular antibodies in mammalian cells. EMBO J. 1: 101−108.
- Werge, T.M., Bioeca, S. and Cattaneo, A. 1991. Intracellular immunization: cloning and intracellular expression of a monoclonal antibody to the p21ras protein. FBBS Lett. 274: 193−198. | Article |
- Hiatt, A., Cafferkey, R. and Bowdish, K. 1989. Production of antibodies in transgenic plants. Nature 342: 76−78. | Article | PubMed | ISI | ChemPort |
- Benvenuto, E., Ordàs, R.J., Tavazza, R., Ancora, G., Biocca, S., Cattaneo, A. and Galeffi, P. 1991. "Phytoantibodies": a general vector for the expression of immunoglobulin domains in transgenic plants. Plant Mol. Biol. 17: 865−874. | PubMed | ISI | ChemPort |
- Barbacid, M. 1987. ras-Genes. Ann. Rev. Biochem. 56: 779−827. | Article | PubMed | ISI | ChemPort |
- Downward, J. 1990. The ras superfamily of small GTP-binding proteins. Trends Biochem. Sci. 15: 469−472. | Article | PubMed | ISI |
- Birchmeier, C., Broek, D. and Wigler, M. 1988. Ras proteins can induce meiosis in Xenopus oocytes. Cell 43: 615−621. | Article |
- Arion, D.L., Meijer, L., Brizuela, L. and Beach, D. 1988. cdc2 is a component of the M phase-specific histone HI kinase: evidence for identity with MPF. Cell 55: 371−378. | Article | PubMed | ISI | ChemPort |
- Furth, M.E., Davis, L.J., Fleurdelys, B. and Scolnick, E.M. 1982. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. J. Virol. 43: 294−304. | PubMed | ISI | ChemPort |
- Mason, J.O., Williams, G.T. and Neuberger, M.S. 1988. The half-life of immunoglobulin mRNA increases during B-cell differentiation: a possible role for targeting to membrane-bound polysomes. Genes and Development 2: 1003−1011. | PubMed | ChemPort |
- Werge, T.M., Bradbury, A.M., Di Luzio, A. and Cattaneo, A. 1992. A recombinant cell line expressing a form of the anti-p21ras antibody. Oncogene 7: 1033−1035. | PubMed | ISI | ChemPort |
- Harlow, E., Crawford, L.V., Pim, D.C. and Williamson, N.M. 1981. Monoclonal antibodies specific for Simian Virus 40 tumor antigens. J. Virol. 39: 861−869. | PubMed | ISI | ChemPort |
- Bradbury, A.M., Persiç, L., Werge, T. and Cattaneo, A. 1993. The use of living columns to select specific phage antibodies. Bio/Technology 11: 1570−1573. | PubMed |
- Biocca, S., Pierandrei-Amaldi, P. and Cattaneo, A. 1993. Intracellular expression of anti-p21ras single chain Fv fragments inhibits meiotic maturation of Xenopus oocytes. Biochem. Biophys. Res. Comm. 197: 422−427. | Article | PubMed | ISI | ChemPort |
- Evan, G.I., Lewis, G.K., Ramsay, G. and Bishop, J.M. 1985. Isolation of monoclonal antibodies specific for human c-mye proto-oncogene product. Mol. Cell. Biol. 5: 3610−3616. | PubMed | ISI | ChemPort |
- Deshpande, A.K. and Kung, H.F. 1987. Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21ras proteins. Mol. Cell. Biol. 7: 1285−1288. | PubMed | ISI | ChemPort |
- Kom, L.J, Siebel, C.W., McCormick, F. and Roth, R.A. 1987. Ras p21 as a potential mediator of insulin action in Xenopus oocytes. Science 236: 840−843. | PubMed | ISI | ChemPort |
- Baltimore, D. 1988. Intracelluiar immunization. Nature 335: 395−396. | Article | PubMed | ISI | ChemPort |
- Marasco, W.A., Haseltine, W.A. and Chen, S. 1993. Design, intracellular expression and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody. Proc. Natl. Acad. Sci. USA 90: 7889−7893. | PubMed | ChemPort |
- Maller, J.L. 1990. Xenopus oocytes and the biochemistry of cell division. Biochemistry 29: 3157−3166. | PubMed | ISI | ChemPort |
- Labib, K. and Nurse, P. 1993. Bring on the phosphatases. Current Biol. 3: 164−166. | ISI | ChemPort |
- Lundgren, K., Walworth, N., Booher, R., Dembski, M., Kirschner, M. and Beach, D. 1991. mikl and weel cooperate in the inhibitory tyrosine phosphorylation of cdc2. Cell 64: 1111−1122. | Article | PubMed | ISI | ChemPort |
- Nebreda, A. R., Martin-Zanca, D., Kaplan, D.R., Parada, L.F. and Santos, E. 1991. Induction by NGF of meiotic maturation of Xenopus oocytes expressing the trk proto-oncogene product. Science 252: 558−561. | PubMed | ISI | ChemPort |
- Winter, G. and Milstein, C. 1991. Man made antibodies. Nature 349: 293−299. | Article | PubMed | ISI | ChemPort |
- Gurdon, J.B. 1976. The Control of Gene Expression in Animal Development, p. 116−126. Harvard University Press, Cambridge, MA. | ChemPort |
- Krieg, P.A. and Melton, D.A. 1987. In vitro RNA synthesis With SP6 RNA polymerase. Methods Enzymol. 155: 397−415. | Article | PubMed | ISI | ChemPort |
- Peculis, B.A.,and Gall, J.G. 1992. Localization of the nucleolar protein N038 in amphibian oocytes. J. Cell Biol. 116: 1−14. | Article | PubMed | ISI | ChemPort |
- Opresko, L.K. and Wiley, H.S. 1990. Functional reconstitutional of the human Epidermal Growth Factor receptor system in Xenopus oocytes. J. Cell Biol. 111: 1661−1671. | Article | PubMed | ISI | ChemPort |
|
