ArticleProceedings of the National Academy of Sciences of the United States of America1999
Selection of antibodies for intracellular function using a two-hybrid in vivo system.
Article in Proceedings of the National Academy of Sciences of the United States of America, 1999. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 57 papers.
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Who cites it
57 citing papers in PubMed, 174 citations in OpenAlex.
- Selection and characterization of human scFvs targeting the SARS-CoV-2 nucleocapsid protein isolated from antibody libraries of COVID-19 patients.Scientific reports · 2024Article
- Acetylation-Specific Interference by Anti-Histone H3K9ac Intrabody Results in Precise Modulation of Gene Expression.International journal of molecular sciences · 2022Article
- Monobodies as tool biologics for accelerating target validation and druggable site discovery.RSC medicinal chemistry · 2021Review
- Antibody Libraries as Tools to Discover Functional Antibodies and Receptor Pleiotropism.International journal of molecular sciences · 2021Review
- Directed evolution in mammalian cells.Nature methods · 2021Review
- A cell-based screening method using an intracellular antibody for discovering small molecules targeting the translocation protein LMO2.Science advances · 2021Article
- Selection and Modelling of a New Single-Domain Intrabody Against TDP-43.Frontiers in molecular biosciences · 2021Article
- Nanobodies Right in the Middle: Intrabodies as Toolbox to Visualize and Modulate Antigens in the Living Cell.Biomolecules · 2020Review
- Exploring cellular biochemistry with nanobodies.The Journal of biological chemistry · 2020Review
- RAS Function in cancer cells: translating membrane biology and biochemistry into new therapeutics.The Biochemical journal · 2020Review
- Tuning GABAergic Inhibition: Gephyrin Molecular Organization and Functions.Neuroscience · 2020Review
- Protein Structural Information and Evolutionary Landscape by In Vitro Evolution.Molecular biology and evolution · 2020Article
- Efficient development and expression of scFv recombinant proteins against PD-L1 surface domain and potency in cancer therapy.Cytotechnology · 2019Article
- Small molecule inhibitors of RAS-effector protein interactions derived using an intracellular antibody fragment.Nature communications · 2018Article
- Intracellular immunization against HIV infection with an intracellular antibody that mimics HIV integrase binding to the cellular LEDGF protein.Scientific reports · 2017Article
- Assessment of antibody library diversity through next generation sequencing and technical error compensation.PloS one · 2017Article
- A novel intracellular antibody against the E6 oncoprotein impairs growth of human papillomavirus 16-positive tumor cells in mouse models.Oncotarget · 2016Article
- Intracellular targeting with engineered proteins.F1000Research · 2016Review
- A novel N-terminal region of the membrane β-hexosyltransferase: its role in secretion of soluble protein by Pichia pastoris.Microbiology (Reading, England) · 2016Article
- LMO2 at 25 years: a paradigm of chromosomal translocation proteins.Open biology · 2015Review
Corrections and comments
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Authors and funding
5 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Expression of antibodies inside cells has been used successfully to ablate protein function. This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences. A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in the cytoplasm. To overcome this problem, we have developed an in vivo assay for functional intracellular antibodies using a two-hybrid approach. In this assay, antibody, as single-chain Fv (scFv) linked to a transcriptional transactivation domain, can interact with a target antigen, linked to a LexA-DNA binding domain, and thereby activate a reporter gene. We find that several characterized antibodies can bind their target antigen in eukaryotic cells in this two-hybrid format, and we have been able to isolate intracellular binders from among sets of scFv that can bind antigen in vitro. Furthermore, we show a model selection in which a single scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate capture of antibody specificities from complex mixtures. The approach can provide the basis for de novo selection of intracellular scFv from libraries, such as those made from spleen RNA after immunization with antigen, for intracellular analysis of protein function based only on genomic or cDNA sequences.
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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.