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Selection for intrabody solubility in mammalian cells using GFP fusions.
Guglielmi L., Denis V., Vezzio-Vié N., Bec N., Dariavach P., Larroque C., Martineau P.
Protein Engineering, Design and Selection 24, 12 (2011) 873-81 - http://www.hal.inserm.fr/inserm-00633286
 (21997307) 
Selection for intrabody solubility in mammalian cells using GFP fusions.
Laurence Guglielmi1, Vincent Denis1, Nadia Vezzio-Vié1, Nicole Bec1, Piona Dariavach1, 2, Christian Larroque1, Pierre Martineau () 1
1 :  IRCM - Institut de recherche en cancérologie de Montpellier
INSERM : U896 – Université Montpellier I – CRLCC Val d'Aurelle - Paul Lamarque
208 rue des Apothicaires F-34298 Montpellier
France
2 :  UM2 - Université Montpellier 2 - Sciences et Techniques
http://www.univ-montp2.fr/
Université Montpellier II - Sciences et techniques – PRES Sud de France
Place Eugène Bataillon - 34095 Montpellier cedex 5
France
Soluble intrabody selection in mammalian cells
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.
Sciences du Vivant/Biochimie, Biologie Moléculaire
Sciences du Vivant/Biologie cellulaire
Anglais
1741-0126

Articles dans des revues avec comité de lecture
10.1093/protein/gzr049
Protein Engineering, Design and Selection (Protein Eng Des Sel)
Publisher Oxford University Press (OUP): Policy B - Oxford Open Option B
ISSN 1741-0126 (eISSN : 1741-0134)
internationale
12/2011
13/10/2011
24
12
873-81

aggregation / degradation / folding / GFP / intrabody
Cell Line – Cytoplasm – Escherichia coli – Green Fluorescent Proteins – Humans – Recombinant Fusion Proteins – Single-Chain Antibodies – Solubility – Tubulin
This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale and the University of Montpellier 1.
Liste des fichiers attachés à ce document : 
DOC
Guglielmi.doc(122 KB)
PDF
figure1.pdf(2.8 MB)
figure2.pdf(286.5 KB)
figure3.pdf(4.8 MB)
figure4.pdf(62.3 KB)
figure5.pdf(125.6 KB)
Guglielmi.pdf(175 KB)
inserm-00633286_edited.pdf(515.5 KB)
ANNEX
supplementary_S1.pdf(333.1 KB)
supplementary_S2.pdf(580.4 KB)
supplementary_S3.pdf(425.6 KB)
XHTML
index.xhtml(70.7 KB)