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Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface.
Open accessAbstract
We have expressed a single chain Fv (scFv) antibody fragment, consisting of the variable heavy and variable light domains from two separate anti-digoxin monoclonal antibodies, on the external surface of Escherichia coli by fusing it to an Lpp-OmpA hybrid previously shown to direct heterologous proteins to the cell surface. This scFv fusion was expressed at a high level and was shown to bind the hapten with high affinity and specificity. Whole cell ELISAs, fluorescence microscopy, protease sensitivity, and flow cytometry all confirmed that the scFv was anchored on the outer membrane and was accessible on the surface. Utilizing fluorescence-activated cell sorting, we were able to specifically enrich scFv-producing cells from a 10(5)-fold excess of control cells in only two steps. The expression of antibody fragments on the surface of E. coli is being evaluated as an attractive method for the in vitro production and selection of useful antibody fragments.
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Citations
Cited by
Yeast surface display for screening combinatorial polypeptide libraries
Boder, E. T., & Wittrup, K. D. (1997) · Nature Biotechnology · doi:10.1038/nbt0697-553 · Cited by 1,760
Stability engineering of antibody single-chain Fv fragments
Wörn, A., & Plückthun, A. (2001) · Journal of Molecular Biology · doi:10.1006/jmbi.2000.4265 · Cited by 616
Antibodies and Genetically Engineered Related Molecules: Production and Purification
Roque, A. C. A., Lowe, C. R., & Taipa, M. A. (2004) · Biotechnology Progress · doi:10.1021/bp030070k · Cited by 360
Müller, S., & Nebe-von-Caron, G. (2010) · FEMS Microbiology Reviews · doi:10.1111/j.1574-6976.2010.00214.x · Cited by 356
The Bacterial Twin-Arginine Translocation Pathway
Lee, P. A., Tullman-Ercek, D., & Georgiou, G. (2006) · Annual Review of Microbiology · doi:10.1146/annurev.micro.60.080805.142212 · Cited by 347
Self-citation
Metadata
- Authors
- Francisco, J. A., Campbell, R., Iverson, B. L., & Georgiou, G.
- Venue
- Proceedings of the National Academy of Sciences
- Published
- 1993-11-15
- Volume
- 90
- Issue
- 22
- Pages
- 10444-10448
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- OpenAlex
- W2024127494
- Cited by
- 251
- References
- 0
- FWCI
- 8.70
- Type
- journal article
- Language
- en
- Open access
- green
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- Francisco, J. A., Campbell, R., Iverson, B. L., & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface. Proceedings of the National Academy of Sciences. https://doi.org/10.1073/pnas.90.22.10444
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Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).
Semantic Scholar’s data is described in: Kinney, R., et al. (2023). The Semantic Scholar Open Data Platform. arXiv:2301.10140.
