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Intracellular expression of an anti-ras single chain Fv

University of British Columbia

The small GTP binding proteins of the Ras superfamily have been implicated in regulating cell growth and differentiation. Much of these data have been generated by the microinjection of the neutralizing antibody to Ras, Y13.259. In studying the role of Ras in mammalian cells I have chosen to attenuate Ras activity by the intracellular expression of a neutralizing anti-Ras single chain Fv (scFv) derived from Y13.259. This approach has the distinct advantage over microinjection in that it permits the study of the biochemical consequence of abrogating Ras activity. The genes for the rat monoclonal, Y13-259, were subcloned and engineered to produce a uni-valent monomeric protein which was shown to recognize the same epitope on Ras (residues 60-76) as the parent antibody molecule. I have successfully expressed scFv-Y13.259 in the SV-40 large-T transformed mouse fibroblast line, SVT-2. Additionally, expression of scFv-Y13.259 did not decrease the half-life of Ras, and the fact that 80-90% of the Ras could be detected complexed with scFv-Y13.259 indicated that this simple method may be useful in uncovering functions of Ras when a given site is masked by the scFv. Furthermore a myristylated version of scFv Y13.259 that localizes to the plasma membrane caused a pronounced change in morphology when expressed in SVT-2 cells. When expressed in the B cell lymphoma line, WEHI-231, results showed that blocking of the Switch II region of Ras by scFv-Y13.259 blocks proliferation and leads to apoptosis. Given that the parental cells respond in the same manner to withdrawal of serum, my results suggest that continued survival and proliferation of this tumor requires a Ras dependant signal from serum. Cell cycle analysis of these scFv-Y13.259 expressing WEHI-231 cells reveals that an apparent block at the G₁ to S phase boundary of the cell cycle was the reason for their eventual demise.

Thesis/Dissertation

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2009-04-15

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http://hdl.handle.net/2429/7172

Genetics

University of British Columbia

UBCV

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