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Cited by 75Protein purification and stabilityMonoclonal and Polyclonal Antibodies ResearchProtein Structure and Dynamics

Evaluation of scFv protein recovery from E. coli by in vitro refolding and mild solubilization process

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Sarker, Animesh, Rathore, Abhishek Singh, Gupta, Rinkoo Devi·2019·Microbial Cell Factories
DOI 10.1186/s12934-019-1053-9PMC PMC6330739
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Abstract

BACKGROUND: The production of therapeutically active single chain variable fragment (scFv) antibody is still challenging in E. coli due to the aggregation propensity of recombinant protein into inclusion bodies (IBs). However, recent advancement of biotechnology has shown substantial recovery of bioactive protein from such insoluble IBs by solubilization and refolding processes. In addition, gene fusion technology has also widely been used to improve the soluble protein production using E. coli. This study demonstrates that mild-solubilization and in vitro refolding strategies, both are capable to recover soluble scFv protein from bacterial IBs, although the degree of success is greatly influenced by different fusion tags with the target protein. RESULTS: It was observed that the most commonly used fusion tag, i.e., maltose binding protein (MBP) was not only influenced the cytoplasmic expression in E. coli but also greatly improved the in vitro refolding yield of scFv protein. On the other hand, mild solubilization process potentially could recover soluble and functional scFv protein from non-classical IBs without assistance of any fusion tag and in vitro refolding step. The recovery yield achieved by mild solubilization process was also found higher than denaturation-refolding method except while scFv was refolded in fusion with MBP tag. Concomitantly, it was also observed that the soluble protein achieved by mild solubilization process was better structured and functionally more active than the one achieved by in vitro refolding method in the absence of MBP tag or refolding enhancer. CONCLUSIONS: Maltose binding protein tagged scFv has shown better refolding and solubility yields as compare to mild solubilization process. However, in terms of cost, time and tag free nature, mild solubilization method for scFv recovery from bacterial IBs is considerable for therapeutic application and further structural studies.

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  • Protein recovery strategies from inclusion bodies via solubilization and refolding processes are laborious, time-consuming and expensive, although various refolding procedures have been developed for therapeutic proteins and applied for antibody fragments.

    “Protein recovery strategies from inclusion bodies via solubilization and refolding processes are laborious, time-consuming and expensive, although various refolding procedures have been developed for therapeutic proteins and applied for antibody fragments [78,79,80].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [78]

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Cited by

Sorted by citations

Authors
Sarker, A., Rathore, A. S., & Gupta, R. D.
Affiliations
South Asian University (IN)
ORCID
Gupta, Rinkoo Devi
Venue
Microbial Cell Factories
Publisher
Springer Science and Business Media LLC
Published
2019-01-14
Volume
18
Issue
1
Pages
5-5
ISSN
1475-2859 (ISSN-L)
DOI
10.1186/s12934-019-1053-9
PMC
PMC6330739
OpenAlex
W2944271242
Cited by
75
References
1
Citations by year
2026: 12 · 2025: 7 · 2024: 8 · 2023: 10 · 2022: 17 · 2021: 10 · 2020: 8 · 2019: 3
FWCI
3.14
Type
journal article
Language
en
Open access
Licence
Locations
  • Microbial Cell Factories (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
South Asian University (Start-up fund)
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 › Protein Structure and Dynamics
Keywords
scFv, scFv antibodies, inclusion bodies, protein refolding, recombinant protein recovery, protein aggregation, therapeutic antibodies, Escherichia coli expression
MeSH terms
Antigen-Antibody Reactions, Inclusion Bodies, Circular Dichroism, Escherichia coli, Protein Denaturation, Recombinant Proteins, Solubility, Single-Chain Antibodies, Maltose-Binding Proteins, Protein Refolding

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