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PEDS Advance Access originally published online on September 30, 2008
Protein Engineering Design and Selection 2009 22(3):149-158; doi:10.1093/protein/gzn053
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

This article appears in the following Protein Engineering issue: Antibody Special Issue [View the issue table of contents]

Antibody library selection by the β-lactamase protein fragment complementation assay

Paola Secco1, Elena D'Agostini1, Roberto Marzari2, Marta Licciulli2, Roberto Di Niro2, Sara D'Angelo1, Andrew R.M. Bradbury3, Umberto Dianzani1, Claudio Santoro1 and Daniele Sblattero1,4

1Department of Medical Sciences, and Interdisciplinary Research Centre on Autoimmune Diseases (IRCAD), University of Eastern Piedmont, Via Solaroli 17, 28100 Novara 2Department of Biology, University of Trieste, Via L. Giorgieri 10, 34127 Trieste, Italy 3B Division, MS-M888, Los Alamos National Laboratory, Los Alamos, NM 87545, USA

4 To whom correspondence should be addressed. E-mail: daniele.sblattero{at}med.unipmn.it

Protein fragment complementation assay (PCA) is based on the interaction between two protein partners (e.g. target antigen and antibody), which are genetically fused to the two halves of a dissected marker protein. Binding of the two partners reassembles the marker protein and hence reconstitutes its activity. In this work we have developed the first application of β-lactamase-based PCA for the isolation of single chain Fv fragments (scFvs) binding to the human receptor RON from a naïve library. Specific scFvs with the ability to immunoprecipitate could be isolated after a single round of PCA selection from an scFv repertoire previously pre-selected by phage display. Furthermore, the PCA was used to successfully map the epitopes recognized by the selected scFvs by screening them against a small library of random RON fragments.

Keywords: β-lactamase/PCA/phage display/scFv

Received August 29, 2008; revised August 29, 2008; accepted September 5, 2008.


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