INTRACELLULAR IMMUNIZATION WITH CYTOSOLIC RECOMBINANT ANTIBODIES

We report the application of a strategy to inactivate cellular protein s in vertebrate cells based on the intracellular expression of immunog lobulin genes. We have selected, in this instance, the p21 protein, en coded by the ras proto-oncogene, as a target protein. The variable reg ions of the neutralizing anti-p21ras monoclonal antibody Y13-259 were cloned in vectors for the expression of either the whole antibody mole cule or its single-chain Fv fragment (ScFv) derivative. In order to ta rget the recombinant antibodies to the cytosol, their hydrophobic lead er 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 ooc yte plasma membrane, and they markedly inhibit the H1 kinase activity induced by insulin. Moreover, cytosolic anti-p21ras ScFv fragments blo ck the ensuing meiotic maturation. Thus the intracellular expression o f both whole antibodies and antibody domains can be used to block a bi ological function.