High.-level expression and characterization of an anti-VEGF165 single-chain variable fragment (scFv) by small ubiquitin-related modifier fusion in Escherichia coli
Open accessAbstract
Antibodies currently constitute the most rapidly growing class of human therapeutics; however, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. High expression of active single-chain antibody fragment (scFv) in Escherichia coli has not been successful, as the protein contains three intramolecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the scFv gene against VEGF165 with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-scFv fusion gene that was highly expressed in the BL21(DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-scFv, was up to 28.5% of the total cellular protein. The fusion protein was purified by Ni nitrilotriacetic acid (NTA) affinity chromatography and cleaved by a SUMO-specific protease to obtain the native scFv, which was further purified by Ni-NTA affinity chromatography. The result of the high-performance liquid chromatography showed that the purity of the recombinant cleaved scFv was greater than 98%. The primary structure of the purified scFv was confirmed by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analysis. In vitro activity assay demonstrated that the recombinant scFv could dose-dependently inhibit VEGF165-induced human umbilical vein-derived endothelial cell proliferation. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant scFv with native sequences.
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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 [121]
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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
Ukkonen, K., Veijola, J., Vasala, A., & Neubauer, P. (2013) · Microbial Cell Factories · doi:10.1186/1475-2859-12-73 · Cited by 65 · see citing works
Rezaie, F., Davami, F., Mansouri, K., Agha Amiri, S., Fazel, R., Mahdian, R., Davoudi, N., Enayati, S., Azizi, M., & Khalaj, V. (2017) · Journal of Applied Microbiology · doi:10.1111/jam.13483 · Cited by 15 · see citing works
- Authors
- Ye, T., Lin, Z., & Lei, H.
- Affiliations
- Lishui University (CN)
- Corresponding author
- Lin, Zhihua
- Venue
- Applied Microbiology and Biotechnology
- Published
- 2008-11
- Volume
- 81
- Issue
- 2
- Pages
- 311-317
- ISSN
- 0175-7598 (ISSN-L)
- 1432-0614
- PMC
- PMC7079844
- OpenAlex
- W2072537268
- Cited by
- 25
- References
- 0
- Citations by year
- 2026: 1 · 2022: 2 · 2020: 1 · 2019: 3 · 2017: 2 · 2016: 2 · 2014: 1 · 2013: 4 · 2012: 4
- FWCI
- 1.28
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Applied Microbiology and Biotechnology (publishedVersion)
- PubMed Central (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 › Molecular Biology › Biochemical and Structural Characterization
- Keywords
- scFv, VEGF165, small ubiquitin-related modifier, SUMO fusion, recombinant antibody production, Escherichia coli expression, intramolecular disulfide bond, endothelial cell proliferation inhibition, Escherichia coli BL21(DE3)
- MeSH terms
- Antibodies, Cells, Cultured, Chromatography, Affinity, Escherichia coli, Humans, Immunoglobulin Variable Region, Recombinant Fusion Proteins, Gene Expression, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Sequence Analysis, Protein, SUMO-1 Protein, Vascular Endothelial Growth Factor A, Endothelial Cells, Cell Proliferation
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