Cited by 18Monoclonal and Polyclonal Antibodies ResearchProtein purification and stabilityBacterial Genetics and Biotechnology

Construction and periplasmic expression of the Anti-EGFRvIII ScFv antibody gene in Escherichia coli

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DEWI, Kartika Sari·2016·Scientia Pharmaceutica

Abstract

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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Crossref · 10.3797/scipharm.isp.2015.06

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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].”

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Research works printed but not linked: 2

  1. [16]Dewi KS Retnoningrum DS Riani C Fuad AM Periplasmic expression of gene encoding anti-EGFRviii single-chain variable fragment antibody using pelb leader sequence in Escherichia coli 2014 11 11–12 Surabaya Proceeding of The 1st International Conference on Pharmaceutics and Pharmaceutical Sciences 24 29
  2. [23]King DJ Secretion of antibody fragments from E. coli. Applications and engineering of monoclonal antibodies 1998 UK CRC Press 170 171

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Authors
DEWI, K. S.
Affiliations
Bandung Institute of Technology (ID)
Corresponding author
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
OpenAlex
W2325599582
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
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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