A plasmid system for optimization of Fab’ production in Escherichia coli: Importance of balance of heavy chain and light chain synthesis
Abstract
We demonstrate the importance of optimizing the balance of light chain (LC) and heavy chain (HC) expression to achieve high level production of Fab' fragments in the Escherichia coli periplasm. The LC:HC balance has been controlled by varying the codon usage of the signal peptide (SP) and 5' mature domain coding regions. Different SP coding regions have been identified from a codon wobble-based library using alkaline phosphatase (AP) as a reporter gene. A plasmid system that enables random combination of these variant SP coding regions is used to construct optimized Fab' expression plasmids. These small plasmid libraries facilitated selection of optimal Fab' expression plasmids and resulted in increases of periplasmic yield, up to 580 mgL(-1) from E. coli fermentations and will enable rapid variable region subcloning and selection of future Fab(') expression plasmids.
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Production of Fab fragments in E. coli is challenging because it is necessary to achieve optimum rather than maximum expression of both chains, requiring the best ratio of HC and LC and protein folding rates, and separately expressed light and heavy chains must assemble correctly with four intrachain and one interchain disulfide bond.
“In addition to the possible aggregation and degradation issues, also the production of Fab fragments in E. coli hosts results challenging. It is indeed necessary to achieve the “optimum” expression rather than the “maximum” expression of both chains because the best ratio of HC and LC and protein folding rates are needed and the separately expressed light and heavy chains must assemble correctly”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [38]
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Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments
Sandomenico, A., Sivaccumar, J. P., & Ruvo, M. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21176324 · Cited by 134 · see citing works
“It is indeed necessary to achieve the “optimum” expression rather than the “maximum” expression of both chains because the best ratio of HC and LC and protein folding rates are needed and the separately expressed light and heavy chains must assemble correctly to constitute the functional heterodimer with four intracha…”
Background
- Authors
- Humphreys, D. P., Carrington, B., Bowering, L. C., Ganesh, R., Sehdev, M., Smith, B. J., King, L. M., Reeks, D. G., Lawson, A., & Popplewell, A. G.
- Corresponding author
- Humphreys, David P
- Venue
- Protein Expression and Purification
- Publisher
- Elsevier BV
- Published
- 2002-11
- Volume
- 26
- Issue
- 2
- Pages
- 309-320
- ISSN
- 1046-5928 (ISSN-L)
- OpenAlex
- W2068450390
- Cited by
- 54
- References
- 0
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 1 · 2023: 2 · 2022: 5 · 2021: 1 · 2020: 2 · 2019: 3 · 2017: 1 · 2015: 2 · 2014: 3 · 2013: 2 · 2012: 5
- FWCI
- 3.14
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Protein Expression and Purification (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Keywords
- Fab fragment, antibody fragments, alkaline phosphatase reporter, plasmid library, recombinant antibody production
- MeSH terms
- Base Sequence, Codon, Enzyme-Linked Immunosorbent Assay, Escherichia coli, Immunoglobulin Fab Fragments, Molecular Sequence Data, Plasmids, Mass Spectrometry, Genes, Reporter
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