tailieunhanh - Báo cáo khoa học: Extended half-life upon binding of destabilized intrabodies allows specific detection of antigen in mammalian cells

The ectopic expression of antibody fragments inside mammalian cells (intrabodies) is a challenging approach for probing and modulating target activities. We previously described the shuttling activity of intracellularly expressedEscherichia colib-galactosidase conferred by the single-chain Fv (scFv) fragment 13R4 equipped with nuclear import⁄export signals. | iFEBS Journal Extended half-life upon binding of destabilized intrabodies allows specific detection of antigen in mammalian cells Annie-Paule Sibler1 Jerome Courtete1 Christian D. Muller2 Gabrielle Zeder-Lutz1 and Etienne Weiss1 1 UMR 700 Ecole Superieure de Biotechnologie de Strasbourg Illkirch France 2 UMR 7034 Faculte de Pharmacie Illkirch France Keywords antigen detection in mammalian cells destabilizing PEST signal sequence functional solubility half-life intrabody Correspondence E. Weiss UMR 7100 Ecole Superieure de Biotechnologie de Strasbourg Boulevard Sebastien Brant BP10413 67412 Illkirch Cedex France Fax 33 390244770 Tel 33 390244767 E-mail eweiss@ Note Annie-Paule Sibler and Jerome Courtete contributed equally to this work. Received 2 February 2005 revised 1 April 2005 accepted 7 April 2005 doi The ectopic expression of antibody fragments inside mammalian cells intrabodies is a challenging approach for probing and modulating target activities. We previously described the shuttling activity of intracellularly expressed Escherichia coli b-galactosidase conferred by the single-chain Fv scFv fragment 13R4 equipped with nuclear import export signals. Here by appending to scFvs the proteolytic PEST signal sequence a protein region rich in proline glutamic acid serine and threonine of mouse ornithine decarboxylase we tested whether short-lived or destabilized intrabodies could affect the steady-state level of target by redirecting it to the protea-somes. In the absence of antigen the half-life of the modified scFv 13R4 relative to untagged molecules was considerably reduced in vivo. However after coexpression with either cytoplasmic or nuclear antigen the destabilized 13R4 fragments were readily maintained in the cell and strictly colocalized with b-galactosidase. Analysis of destabilized site-directed mutants that were as soluble as 13R4 in the intracellular context demonstrated that binding to antigen was .

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