title: Selection for intrabody solubility in mammalian cells using GFP fusions.
abstract: Single-chain antibody fragments (scFv) expressed in the cytoplasm of mammalian cells, also called intrabodies, have many applications in functional proteomics. These applications are, however, limited by the aggregation-prone behaviour of many intrabodies. We show here that two scFv with highly homologous sequences and comparable soluble expression levels in Escherichia coli cytoplasm have different behaviours in mammalian cells. When over-expressed, one of the scFv aggregates in the cytoplasm whereas the second one is soluble and active. When expressed at low levels, using a retroviral vector, as a fusion with the green fluorescent protein (GFP) the former does not form aggregates and is degraded, resulting in weakly fluorescent cells, whereas the latter is expressed as a soluble protein, resulting in strongly fluorescent cells. These data suggest that the GFP signal can be used to evaluate the soluble expression of intrabodies in mammalian cells. When applied to a subset of an E.coli-optimised intrabody library, we showed that the population of GFP+ cells contains indeed soluble mammalian intrabodies. Altogether, our data demonstrate that the requirements for soluble intrabody expression are different in E.coli and mammalian cells, and that intrabody libraries can be directly optimised in human cells using a simple GFP-based assay.
authors: IRCM,  Institut de Recherche en Cancérologie de Montpellier, Montpellier F-34298,  France.
authors: Laurence
authors: L
authors: Guglielmi
authors: Vincent
authors: V
authors: Denis
authors: Nadia
authors: N
authors: Vezzio-Vié
authors: Nicole
authors: N
authors: Bec
authors: Piona
authors: P
authors: Dariavach
authors: Christian
authors: C
authors: Larroque
authors: Pierre
authors: P
authors: Martineau
mesh_terms: D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000302:isolation & purification*; D012995:Solubility; D014404:Tubulin / Q000276:immunology
chemical_list: D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D014404:Tubulin; D049452:Green Fluorescent Proteins