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 expressed Escherichia coli beta-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 proteasomes. 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 beta-galactosidase. Analysis of destabilized site-directed mutants, that were as soluble as 13R4 in the intracellular context, demonstrated that binding to antigen was essential for survival under these conditions. This unique property allowed specific detection of beta-galactosidase, even when expressed at low level in stably transformed cells, and permitted isolation by flow cytometry from a transfected cell mixture of those living cells specifically labeled with bound intrabody. Altogether, we show that PEST-tagged intrabodies of sufficient affinity and solubility are powerful tools for imaging the presence and likely the dynamics of protein antigens that are resistant to proteasomal degradation in animal cells.
Sibler
Annie-Paule
AP
UMR 700, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France.
Courtête
Jérôme
J
Muller
Christian D
CD
Zeder-Lutz
Gabrielle
G
Weiss
Etienne
E
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D003593:Cytoplasm; D005434:Flow Cytometry; D006207:Half-Life; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000276:immunology / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D051379:Mice; D009856:Oncogene Proteins, Viral / Q000235:genetics; D009955:Ornithine Decarboxylase / Q000276:immunology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D021381:Protein Transport / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics; D001616:beta-Galactosidase / Q000276:immunology / Q000378:metabolism
C068651:E6 protein, Human papillomavirus type 16; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; D001616:beta-Galactosidase; D046988:Proteasome Endopeptidase Complex; D009955:Ornithine Decarboxylase