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

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Basic-Leucine Zipper Transcription Factors / chemistry
  • Basic-Leucine Zipper Transcription Factors / genetics
  • Basic-Leucine Zipper Transcription Factors / metabolism
  • Directed Molecular Evolution / methods*
  • Escherichia coli / genetics
  • Escherichia coli Proteins / genetics
  • Intracellular Space / chemistry
  • Intracellular Space / metabolism
  • Membrane Transport Proteins / genetics
  • Periplasm / chemistry
  • Periplasm / metabolism
  • Protein Binding
  • Protein Engineering / methods*
  • Protein Folding
  • Protein Stability
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Single-Chain Antibodies / chemistry
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / metabolism*
  • alpha-Synuclein / chemistry
  • alpha-Synuclein / metabolism


  • Basic-Leucine Zipper Transcription Factors
  • Escherichia coli Proteins
  • GCN4 protein, S cerevisiae
  • Membrane Transport Proteins
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Single-Chain Antibodies
  • alpha-Synuclein
  • twin-arginine translocase complex, E coli