ArticleProceedings of the National Academy of Sciences of the United States of America2009
Versatile selection technology for intracellular protein-protein interactions mediated by a unique bacterial hitchhiker transport mechanism.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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Who cites it
19 citing papers in PubMed, 39 citations in OpenAlex.
- Modified protein selection strategy based on Escherichia coli's Hitchhiker transport and validation through selection of nanobodies targeting bovine interferon gamma.Scientific reports · 2026Article
- Isolation of Antigen-Specific Nanobodies From Synthetic Libraries Using a Protein Selection Strategy That Combines MACS-Based Screening of YSD and FLI-TRAP.Bio-protocol · 2026Article
- Isolation of PCSK9-specific nanobodies from synthetic libraries using a combined protein selection strategy.Scientific reports · 2025Article
- Target-dependent RNA polymerase as universal platform for gene expression control in response to intracellular molecules.Nature communications · 2023Article
- Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli.Nature communications · 2023Article
- A survival selection strategy for engineering synthetic binding proteins that specifically recognize post-translationally phosphorylated proteins.Nature communications · 2019Article
- Selection of Protein-Protein Interactions of Desired Affinities with a Bandpass Circuit.Journal of molecular biology · 2019Article
- Computational affinity maturation of camelid single-domain intrabodies against the nonamyloid component of alpha-synuclein.Scientific reports · 2018Article
- Directed evolution to improve protein folding in vivo.Current opinion in structural biology · 2018Review
- An Intrabody Drug (rAAV6-INT41) Reduces the Binding of N-Terminal Huntingtin Fragment(s) to DNA to Basal Levels in PC12 Cells and Delays Cognitive Loss in the R6/2 Animal Model.Journal of neurodegenerative diseases · 2016Article
- Review
- An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism.Scientific reports · 2014Article
- Optimizing recombinant antibodies for intracellular function using hitchhiker-mediated survival selection.Protein engineering, design & selection : PEDS · 2014Article
- Ubiquibodies, synthetic E3 ubiquitin ligases endowed with unnatural substrate specificity for targeted protein silencing.The Journal of biological chemistry · 2014Article
- Diversity in genetic in vivo methods for protein-protein interaction studies: from the yeast two-hybrid system to the mammalian split-luciferase system.Microbiology and molecular biology reviews : MMBR · 2012Review
- Engineering antibody fitness and function using membrane-anchored display of correctly folded proteins.Journal of molecular biology · 2012Article
- Visualizing interactions along the Escherichia coli twin-arginine translocation pathway using protein fragment complementation.PloS one · 2010Article
- Mining mammalian genomes for folding competent proteins using Tat-dependent genetic selection in Escherichia coli.Protein science : a publication of the Protein Society · 2009Article
- Review
Corrections and comments
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
Abstract
We have developed a reliable genetic selection strategy for isolating interacting proteins based on the "hitchhiker" mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway. This method, designated FLI-TRAP (functional ligand-binding identification by Tat-based recognition of associating proteins), is based on the unique ability of the Tat system to efficiently cotranslocate noncovalent complexes of 2 folded polypeptides. In the FLI-TRAP assay, the protein to be screened for interactions is engineered with an N-terminal Tat signal peptide, whereas the known or putative partner protein is fused to mature TEM-1 beta-lactamase (Bla). Using a series of c-Jun and c-Fos leucine zipper (JunLZ and FosLZ) variants of known affinities, we observed that only those chimeras that expressed well and interacted strongly in the cytoplasm were able to colocalize Bla into the periplasm and confer beta-lactam antibiotic resistance to cells. Likewise, the assay was able to efficiently detect interactions between intracellular single-chain Fv (scFv) antibodies and their cognate antigens. The utility of FLI-TRAP was then demonstrated through random library selections of amino acid substitutions that restored (i) heterodimerization to a noninteracting FosLZ variant, and (ii) antigen binding to a low-affinity scFv antibody. Because Tat substrates must be correctly folded before transport, FLI-TRAP favors the identification of soluble, nonaggregating, protease-resistant protein pairs and, thus, provides a powerful tool for routine selection of interacting partners (e.g., antibody-antigen), without the need for purification or immobilization of the binding target.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.