ArticleProceedings of the National Academy of Sciences of the United States of America2007
APEx 2-hybrid, a quantitative protein-protein interaction assay for antibody discovery and engineering.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2007. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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Who cites it
11 citing papers in PubMed, 51 citations in OpenAlex.
- Disulfide-compatible phage-assisted continuous evolution in the periplasmic space.Nature communications · 2021Article
- A Quantitative Tri-fluorescent Yeast Two-hybrid System: From Flow Cytometry toMolecular & cellular proteomics : MCP · 2020Article
- Protein engineering approaches for antibody fragments: directed evolution and rational design approaches.Turkish journal of biology = Turk biyoloji dergisi · 2019Article
- A single mouse monoclonal antibody, E58 modulates multiple IgE epitopes on group 1 cedar pollen allergens.Molecular immunology · 2016Article
- Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS).PloS one · 2014Article
- Single-cell characterization of autotransporter-mediated Escherichia coli surface display of disulfide bond-containing proteins.The Journal of biological chemistry · 2012Article
- 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
- Article
- Yeast surface two-hybrid for quantitative in vivo detection of protein-protein interactions via the secretory pathway.The Journal of biological chemistry · 2009Article
- Engineering antibody fragments to fold in the absence of disulfide bonds.Protein science : a publication of the Protein Society · 2009Article
- Article
Corrections and comments
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Authors and funding
4 authors at 2 institutions in 3 countries.
Funding
Abstract
We have developed a bacterial system for the discovery of interacting proteins that, unlike other two-hybrid technologies, allows for the selection of protein pairs on the basis of affinity or expression. This technology relies on the anchored periplasmic expression (APEx) of one protein (bait) on the periplasmic side of the inner membrane of Escherichia coli and its interacting partner (prey) as a soluble, epitope-tagged, periplasmic protein. Upon removal of the outer membrane by spheroplasting, periplasmic proteins, including any unbound epitope-tagged prey, are released into the extracellular fluid. However, if the epitope-tagged prey can bind to the membrane-anchored bait, it remains associated with the cell and can be detected quantitatively by using fluorescent anti-epitope tag antibodies. Cells expressing prey:bait pairs exhibiting different affinities can be readily distinguished by flow cytometry. The utility of this technology, called APEx two-hybrid, was demonstrated in two demanding antibody engineering applications: First, single-chain variable fragment (scFvs) with increased affinity to the protective antigen of Bacillus anthracis were isolated from cells coexpressing libraries of scFv random mutants, together with endogenously expressed antigen. Second, APEx two-hybrid coupled with multicolor FACS analysis to account for protein expression was used for the selection of mutant Fab antibody fragments exhibiting improved expression in the bacterial periplasm.
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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.