Construction and periplasmic expression of the Anti-EGFRvIII ScFv antibody gene in Escherichia coli
Open accessAbstract
In the previous study, we constructed an expression vector carrying the anti-EGFRvIII scFv antibody gene with VH-linker-VL orientation. The proteins were successfully produced in the periplasmic space of Escherichia coli. In this study, we substituted the inserted DNA with VL-linker-VH orientation of the anti-EGFRvIII scFv gene and analyzed its expression in E. coli. The DNA fragment was amplified from its cloning vector (pTz-rscFv), subsequently cloned into a previous expression vector containing the pelB signal sequence and his-tag, and then transformed into E. coli TOP10. The recombinant plasmids were characterized by restriction, PCR, and DNA sequencing analyses. The new anti-EGFRvIII scFv antibody proteins have been successfully expressed in the periplasmic compartment of E. coli Nico21(DE3) using 0.1 mM final concentration of IPTG induction. Total proteins, soluble periplasmic and cytoplasmic proteins, solubilized inclusion bodies, and extracellular proteins were analyzed by SDS-PAGE and Western Blot analyses. The results showed that soluble scFv proteins were found in all fractions except from the cytoplasmic space.
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The 22 amino acids long PelB signal sequence from Erwinia carotovora is the most frequently used for transportation of Fabs and scFvs.
“The 22 amino acids long PelB (pectate lyase B) signal sequence from Erwinia carotovora [157] is the most frequently used for transportation of the Fabs [88,96] and scFvs [30,101,158,159].”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [30]
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- Authors
- DEWI, K. S.
- Affiliations
- Bandung Institute of Technology (ID)
- Corresponding author
- DEWI, Kartika Sari
- ORCID
- DEWI, Kartika Sari
- Venue
- Scientia Pharmaceutica
- Publisher
- MDPI AG
- Published
- 2016
- Volume
- 84
- Issue
- 1
- Pages
- 141-152
- ISSN
- 0036-8709 (ISSN-L)
- 2218-0532
- PMC
- PMC4839555
- OpenAlex
- W2325599582
- Semantic Scholar
- c910bc19d93562f064f3b3409e6a7cb11de1eeeb
- Cited by
- 18
- References
- 29
- Citations by year
- 2026: 1 · 2025: 3 · 2023: 3 · 2022: 1 · 2021: 2 · 2020: 3 · 2019: 3 · 2017: 1 · 2016: 1
- FWCI
- 1.45
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by-nc-nd
- Locations
- Scientia Pharmaceutica (publishedVersion, cc-by-nc-nd)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- 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
- EGFRvIII, single-chain antibody, scFv, periplasmic expression, Escherichia coli, recombinant protein expression, inclusion bodies
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