title: Optimizing recombinant antibodies for intracellular function using hitchhiker-mediated survival selection.
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.
authors.affiliation: 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.
authors.forename: Dujduan
authors.initials: D
authors.lastname: Waraho-Zhmayev
authors.affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Bunyarit
authors.initials: B
authors.lastname: Meksiriporn
authors.affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Alyse D
authors.initials: AD
authors.lastname: Portnoff
authors.affiliation: 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.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: DeLisa
mesh_terms: D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D042541:Intracellular Space / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics; D019897:Periplasm / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051844:alpha-Synuclein / Q000737:chemistry / Q000378:metabolism
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D029968:Escherichia coli Proteins; C501391:GCN4 protein, S cerevisiae; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D057127:Single-Chain Antibodies; D051844:alpha-Synuclein; C408585:twin-arginine translocase complex, E coli
keywords: antigen-binding affinity; directed evolution; intracellular antibody engineering; protein folding and stability; twin-arginine translocation