Cytosolic expression of functional Fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase® cultivation mode
Abstract
Aims The Escherichia coli expression system is highly effective in producing recombinant proteins. However, there are some limitations in this system, especially in obtaining correctly folded forms of some complex proteins such as Fab fragments. To improve the solubility and folding quality of Fab fragments, we have examined the effect of simultaneous application of a SUMO fusion tag, EnBase ® cultivation mode and a redox mutant strain in the E. coli expression system. Methods and results A bicistronic gene construct was designed to express an antivascular endothelial growth factor (VEGF) Fab fragment as a model system. The construct contained a dual SUMO fusion gene fragment to encode SUMO-tagged heavy and light chains. While the expression of the construct in batch cultures of BL21 or SHuffle ® transformants produced insoluble and unfolded products, the induction of the transformants in EnBase ® medium resulted in soluble and correctly folded Fab fragment, reaching as high as 19% of the total protein in shuffle strain. The functional assays indicated that the biological activity of the target Fab is similar to the commercial anti-VEGF, Lucentis ® . Conclusions This study demonstrated that the combination of SUMO fusion technology, EnBase ® cultivation system and recruiting a redox mutant of E. coli can efficiently enhance the solubility and productivity of recombinant Fab fragments. Significance and the impact of the study The presented strategy provides not only a novel me…
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SUMO fusion technology has been successfully applied to cytosolic production of Fabs in E. coli; the highest yield of correctly folded and biologically active SUMO-tagged Fab, 12 mg/L, was recovered from cells harvested after 16 h growth at 30 °C post-induction with 0.5 mM IPTG using SHuffle strain and EnBase medium.
“The SUMO fusion technology has been successfully applied to the cytosolic production of Fabs in E. coli. The highest yield of correctly folded and biologically active SUMO-tagged Fab, 12 mg/L, was recovered from cells harvested after a 16 h growth at 30 °C post-induction with 0.5 mM IPTG using the SHuffle strain and the EnBase medium [87].”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [87]
Use of fusion partners such as GST, MBP, SUMO and Trx to target proteins, especially scFv fragments, results in solubility-enhancing properties and increased yields of soluble and active products.
“Several reports have also suggested that the use of fusion partners such as GST [118], maltose-binding protein (MBP) [119,120], small ubiquitin-related modifier (SUMO) [87,121] and thioredoxin (Trx) [122] to the target proteins, especially to scFv fragments, results in solubility-enhancing properties and in increased yields of soluble and active products.”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [87]
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- Authors
- Rezaie, F., Davami, F., Mansouri, K., Agha Amiri, S., Fazel, R., Mahdian, R., Davoudi, N., Enayati, S., Azizi, M., & Khalaj, V.
- Affiliations
- Pasteur Institute of Iran (IR)
- Kermanshah University of Medical Sciences (IR)
- Medical Biotechnology Department; Biotechnology Research Center; Pasteur Institute of Iran; Tehran Iran
- Corresponding author
- Khalaj, V.
- Venue
- Journal of Applied Microbiology
- Publisher
- Oxford University Press (OUP)
- Published
- 2017-06-09
- Volume
- 123
- Issue
- 1
- Pages
- 134-144
- ISSN
- 1364-5072 (ISSN-L)
- OpenAlex
- W2613436790
- Cited by
- 15
- References
- 41
- Citations by year
- 2026: 1 · 2024: 1 · 2022: 2 · 2021: 4 · 2020: 3 · 2019: 2 · 2018: 2
- FWCI
- 0.78
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Applied Microbiology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Pasteur Institute of Iran
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
- Keywords
- Fab fragment, SUMO fusion tag, anti-VEGF antibody, recombinant protein solubility, protein folding, antibody fragments
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