ArticleJournal of the American Chemical Society2014
Engineering bacterial transcription regulation to create a synthetic in vitro two-hybrid system for protein interaction assays.
Article in Journal of the American Chemical Society, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
What it found
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Who cites it
13 citing papers in PubMed, 18 citations in OpenAlex.
- CF2H: a cell-free two-hybrid platform for rapid protein binder screening.Nature communications · 2026Article
- Cell-Free Gene Expression: Methods and Applications.Chemical reviews · 2025Review
- Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing.Nature communications · 2022Article
- Rapid isolation of antigen-specific B-cells using droplet microfluidics.RSC advances · 2020Article
- Microfluidic approaches for the analysis of protein-protein interactions in solution.Biophysical reviews · 2020Review
- Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology.Frontiers in bioengineering and biotechnology · 2020Review
- Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria.Nature communications · 2019Article
- A bacterial three-hybrid assay detects Escherichia coli Hfq-sRNA interactions in vivo.Nucleic acids research · 2018Article
- A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders.Scientific reports · 2016Article
- Article
- Label-free single-cell protein quantification using a drop-based mix-and-read system.Scientific reports · 2015Article
- High-throughput preparation methods of crude extract for robust cell-free protein synthesis.Scientific reports · 2015Article
- Building protein networks in synthetic systems from the bottom-up.Biotechnology advancesReview
Corrections and comments
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Authors and funding
6 authors at 3 institutions in 3 countries.
Funding
Abstract
Transcriptional activation of σ(54)-RNA polymerase holoenzyme (σ(54)-RNAP) in bacteria is dependent on a cis-acting DNA element (bacterial enhancer), which recruits the bacterial enhancer-binding protein to contact the holoenzyme via DNA looping. Using a constructive synthetic biology approach, we recapitulated such process of transcriptional activation by recruitment in a reconstituted cell-free system, assembled entirely from a defined number of purified components. We further engineered the bacterial enhancer-binding protein PspF to create an in vitro two-hybrid system (IVT2H), capable of carrying out gene regulation in response to expressed protein interactions. Compared with genetic systems and other in vitro methods, IVT2H not only allows detection of different types of protein interactions in just a few hours without involving cells but also provides a general correlation of the relative binding strength of the protein interaction with the IVT2H signal. Due to its reconstituted nature, IVT2H provides a biochemical assay platform with a clean and defined background. We demonstrated the proof-of-concept of using IVT2H as an alternative assay for high throughput screening of small-molecule inhibitors of protein-protein interaction.
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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.