ArticleJournal of molecular biology2009
Efficient isolation of soluble intracellular single-chain antibodies using the twin-arginine translocation machinery.
Article in Journal of molecular biology, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 25 papers.
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Who cites it
25 citing papers in PubMed, 53 citations in OpenAlex.
- Two-Tiered Selection and Screening Strategy to Increase Functional Enzyme Production in E. coli.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology.BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy · 2020Review
- 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
- Antibodies inside of a cell can change its outside: Can intrabodies provide a new therapeutic paradigm?Computational and structural biotechnology journal · 2016Review
- Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells.Journal of molecular biology · 2015Article
- Review
- Optimizing recombinant antibodies for intracellular function using hitchhiker-mediated survival selection.Protein engineering, design & selection : PEDS · 2014Article
- Directed evolution of human heavy chain variable domain (VH) using in vivo protein fitness filter.PloS one · 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
- Single-chain fragment variable passive immunotherapies for neurodegenerative diseases.International journal of molecular sciences · 2013Review
- Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels.Microbial cell factories · 2013Article
- Analysis of the relationship between end-to-end distance and activity of single-chain antibody against colorectal carcinoma.Theoretical biology & medical modelling · 2012Article
- The ribosomal exit tunnel as a target for optimizing protein expression in Escherichia coli.Biotechnology journal · 2012Article
- Engineering antibody fitness and function using membrane-anchored display of correctly folded proteins.Journal of molecular biology · 2012Article
- Strain engineering for improved expression of recombinant proteins in bacteria.Microbial cell factories · 2011Review
- Bioinformatics-led design of single-chain antibody molecules targeting DNA sequences for retinoblastoma.International journal of ophthalmology · 2011Article
- Physico-chemical determinants of soluble intrabody expression in mammalian cell cytoplasm.Protein engineering, design & selection : PEDS · 2010Article
- A rapid protein folding assay for the bacterial periplasm.Protein science : a publication of the Protein Society · 2010Article
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
Abstract
One of the most commonly used recombinant antibody formats is the single-chain variable fragment (scFv) that consists of the antibody variable heavy chain connected to the variable light chain by a flexible linker. Since disulfide bonds are often necessary for scFv folding, it can be challenging to express scFvs in the reducing environment of the cytosol. Thus, we sought to develop a method for antigen-independent selection of scFvs that are stable in the reducing cytosol of bacteria. To this end, we applied a recently developed genetic selection for protein folding and solubility based on the quality control feature of the Escherichia coli twin-arginine translocation (Tat) pathway. This selection employs a tripartite sandwich fusion of a protein-of-interest with an N-terminal Tat-specific signal peptide and C-terminal TEM1 beta-lactamase, thereby coupling antibiotic resistance with Tat pathway export. Here, we adapted this assay to develop intrabody selection after Tat export (ISELATE), a high-throughput selection strategy for the identification of solubility-enhanced scFv sequences. Using ISELATE for three rounds of laboratory evolution, it was possible to evolve a soluble scFv from an insoluble parental sequence. We show also that ISELATE enables focusing of an scFv library in soluble sequence space before functional screening and thus can be used to increase the likelihood of finding functional intrabodies. Finally, the technique was used to screen a large repertoire of naïve scFvs for clones that conferred significant levels of soluble accumulation. Our results reveal that the Tat quality control mechanism can be harnessed for molecular evolution of scFvs that are soluble in the reducing cytoplasm of E. coli.
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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.