ArticleProtein engineering, design & selection : PEDS2011
Selection for intrabody solubility in mammalian cells using GFP fusions.
Article in Protein engineering, design & selection : PEDS, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed, 22 citations in OpenAlex.
- Hypervariable loop profiling decodes sequence determinants of antibody stability.Nature structural & molecular biology · 2026Article
- Development of a synthetic library of humanized nanobodies for targeted IL-6 inhibition.Frontiers in bioengineering and biotechnology · 2024Article
- Targeted Intracellular Delivery of Antibodies: The State of the Art.Frontiers in pharmacology · 2018Review
- A Link between Integral Membrane Protein Expression and Simulated Integration Efficiency.Cell reports · 2016Article
- Article
- Specific GFP-binding artificial proteins (αRep): a new tool for in vitro to live cell applications.Bioscience reports · 2015Article
- Review
- Suppressing mutation-induced protein aggregation in mammalian cells by mutating residues significantly displaced upon the original mutation.Biochemical engineering journal · 2014Article
- In-cell intrabody selection from a diverse human library identifies C12orf4 protein as a new player in rodent mast cell degranulation.PloS one · 2014Article
- Article
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Authors and funding
7 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Single-chain antibody fragments (scFv) expressed in the cytoplasm of mammalian cells, also called intrabodies, have many applications in functional proteomics. These applications are, however, limited by the aggregation-prone behaviour of many intrabodies. We show here that two scFv with highly homologous sequences and comparable soluble expression levels in Escherichia coli cytoplasm have different behaviours in mammalian cells. When over-expressed, one of the scFv aggregates in the cytoplasm whereas the second one is soluble and active. When expressed at low levels, using a retroviral vector, as a fusion with the green fluorescent protein (GFP) the former does not form aggregates and is degraded, resulting in weakly fluorescent cells, whereas the latter is expressed as a soluble protein, resulting in strongly fluorescent cells. These data suggest that the GFP signal can be used to evaluate the soluble expression of intrabodies in mammalian cells. When applied to a subset of an E.coli-optimised intrabody library, we showed that the population of GFP+ cells contains indeed soluble mammalian intrabodies. Altogether, our data demonstrate that the requirements for soluble intrabody expression are different in E.coli and mammalian cells, and that intrabody libraries can be directly optimised in human cells using a simple GFP-based assay.
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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.