A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm.
BACKGROUND
Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.


RESULTS
We describe the construction and validation of a large synthetic human single chain antibody fragment library based on a unique framework and optimized for cytoplasmic expression. Focusing the library by mimicking the natural diversity of CDR3 loops ensured that the scFvs were fully human and functional. We show that the library is highly diverse and functional since it has been possible to isolate by phage-display several strong binders against the five proteins tested in this study, the Syk and Aurora-A protein kinases, the alphabeta tubulin dimer, the papillomavirus E6 protein and the core histones. Some of the selected scFvs are expressed at an exceptional high level in the bacterial cytoplasm, allowing the purification of 1 mg of active scFv from only 20 ml of culture. Finally, we show that after three rounds of selection against core histones, more than half of the selected scFvs were active when expressed in vivo in human cells since they were essentially localized in the nucleus.


CONCLUSIONS
This new library is a promising tool not only for an easy and large-scale selection of functional intrabodies but also for the isolation of highly expressed scFvs that could be used in numerous biotechnological and therapeutic applications.
Philibert
Pascal
P
CNRS, UMR5160, CRLC, 15, av, Charles Flahault, BP14491, 34093, Montpellier Cedex 5, France. pascal.philibert@tiscali.fr
Stoessel
Audrey
A
Wang
Wei
W
Sibler
Annie-Paule
AP
Bec
Nicole
N
Larroque
Christian
C
Saven
Jeffery G
JG
Courtête
Jérôme
J
Weiss
Etienne
E
Martineau
Pierre
P
D000818:Animals; D000911:Antibodies, Monoclonal; D000916:Antibody Diversity / Q000235:genetics; D000918:Antibody Specificity / Q000235:genetics; D064127:Aurora Kinases; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D003593:Cytoplasm / Q000235:genetics* / Q000276:immunology*; D015723:Gene Library; D006801:Humans; D050440:Immunoglobulin Subunits / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000276:immunology; D009856:Oncogene Proteins, Viral / Q000276:immunology; D019151:Peptide Library; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000276:immunology; D011505:Protein-Tyrosine Kinases / Q000276:immunology; D040901:Proteomics / Q000379:methods; D012097:Repressor Proteins / Q000276:immunology; D000072377:Syk Kinase; D014404:Tubulin / Q000276:immunology
D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; C068651:E6 protein, Human papillomavirus type 16; D050440:Immunoglobulin Subunits; D047908:Intracellular Signaling Peptides and Proteins; D009856:Oncogene Proteins, Viral; D019151:Peptide Library; D012097:Repressor Proteins; D014404:Tubulin; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases