Optimizing recombinant antibodies for intracellular function using hitchhiker-mediated survival selection
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Publication Details
Author list: Waraho-Zhmayev D., Meksiriporn B., Portnoff A.D., DeLisa M.P., Bradbury A.
Publisher: Oxford University Press
Publication year: 2014
Volume number: 27
Issue number: 10
Start page: 351
End page: 358
Number of pages: 8
ISSN: 1741-0126
eISSN: 1741-0134
Languages: English-Great Britain (EN-GB)
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Abstract
The 'hitchhiker' mechanism of the bacterial twin-arginine translocation pathway has previously been adapted as a genetic selection for detecting pairwise protein interactions in the cytoplasm of living Escherichia coli cells. Here, we extended this method, called FLI-TRAP, for rapid isolation of intracellular antibodies (intrabodies) in the single-chain Fv format that possess superior traits simply by demanding bacterial growth on high concentrations of antibiotic. Following just a single round of survival-based enrichment using FLI-TRAP, variants of an intrabody against the dimerization domain of the yeast Gcn4p transcription factor were isolated having significantly greater intracellular stability that translated to yield enhancements of >10-fold. Likewise, an intrabody specific for the non-amyloid component region of a-synuclein was isolated that has ∼8-fold improved antigen-binding affinity. Collectively, our results illustrate the potential of the FLI-TRAP method for intracellular stabilization and affinity maturation of intrabodies, all without the need for purification or immobilization of the antigen. © The Author 2014. Published by Oxford University Press. All rights reserved.
Keywords
Antigen-binding affinity, Intracellular antibody engineering, Protein folding and stability, Twin-arginine translocation
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