AI Article Synopsis
- Researchers developed a method to inactivate cellular proteins in vertebrate cells by using antibodies, specifically targeting the p21 protein from the ras proto-oncogene.
- They cloned variable regions of a monoclonal antibody to create forms that can be expressed inside cells, modifying them to ensure they stay within the cytosol rather than being secreted.
- In experiments with Xenopus laevis oocytes, these antibodies effectively inhibited a specific kinase activity and blocked meiotic maturation, demonstrating the potential for using antibodies to interrupt biological functions inside cells.
Article Abstract
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 proto-oncogene, 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 single-chain 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.
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| http://dx.doi.org/10.1038/nbt0494-396 | DOI Listing |
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