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Protein Eng Des Sel. 2014 Oct;27(10):351-8. doi: 10.1093/protein/gzu038. Epub 2014 Sep 14.

Optimizing recombinant antibodies for intracellular function using hitchhiker-mediated survival selection.

Author information

1
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA Biological Engineering Program, Faculty of Engineering, King Mongkut's University of Technology Thonburi, 126 Pracha-utid Road, Bangmod, Toongkru, Bangkok 10140, Thailand.
2
Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA.
3
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA md255@cornell.edu.

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 α-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.

KEYWORDS:

antigen-binding affinity; directed evolution; intracellular antibody engineering; protein folding and stability; twin-arginine translocation

PMID:
25225416
PMCID:
PMC4191445
DOI:
10.1093/protein/gzu038
[Indexed for MEDLINE]
Free PMC Article

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