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Cited by 137Monoclonal and Polyclonal Antibodies ResearchProtein purification and stabilityBiochemical and Structural Characterization

Recombinant expression of nanobodies and nanobody-derived immunoreagents

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de Marco, Ario·2020·Protein Expression and Purification
DOI 10.1016/j.pep.2020.105645PMC PMC7151424
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Abstract

Antibody fragments for which the sequence is available are suitable for straightforward engineering and expression in both eukaryotic and prokaryotic systems. When produced as fusions with convenient tags, they become reagents which pair their selective binding capacity to an orthogonal function. Several kinds of immunoreagents composed by nanobodies and either large proteins or short sequences have been designed for providing inexpensive ready-to-use biological tools. The possibility to choose among alternative expression strategies is critical because the fusion moieties might require specific conditions for correct folding or post-translational modifications. In the case of nanobody production, the trend is towards simpler but reliable (bacterial) methods that can substitute for more cumbersome processes requiring the use of eukaryotic systems. The use of these will not disappear, but will be restricted to those cases in which the final immunoconstructs must have features that cannot be obtained in prokaryotic cells. At the same time, bacterial expression has evolved from the conventional procedure which considered exclusively the nanobody and nanobody-fusion accumulation in the periplasm. Several reports show the advantage of cytoplasmic expression, surface-display and secretion for at least some applications. Finally, there is an increasing interest to use as a model the short nanobody sequence for the development of in silico methodologies aimed at optimizing the yields, stability and affinity of recombinant antibodies.

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  • Camelid VHH and shark V-NAR domains are used as templates to generate highly efficient and stable nanobodies.

    “Given their high-affinity, solubility and stability also in absence of the partner VL domain, the camelid VH domains (VHH) [4] and the shark VH domains called V-NAR (New Antigen Receptor) [5], are currently used as templates to generate highly efficient and stable new antibody fragments collectively termed nanobodies [6,7,8].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [7]

    The Introduction (page unavailable) identifies camelid VHH domains as nanobodies and describes their binding affinity, structural stability, and engineering into reagents. It does not support the claim that shark V-NAR domains are also used as templates for nanobodies; searches for V-NAR and shark yielded no relevant passage. (LLM, conf=0.87)

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Cited by

Sorted by citations

Authors
de Marco, A.
Venue
Protein Expression and Purification
Publisher
Elsevier BV
Published
2020-08
Volume
172
Pages
105645
ISSN
1046-5928 (ISSN-L)
DOI
10.1016/j.pep.2020.105645
PMC
PMC7151424
OpenAlex
W3015574295
Cited by
137
References
3
Citations by year
2026: 23 · 2025: 27 · 2024: 16 · 2023: 29 · 2022: 18 · 2021: 14 · 2020: 7 · 2019: 3
FWCI
2.51
Type
journal article
Language
en
Open access
Licence
Locations
  • Protein Expression and Purification (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • RUNG (submittedVersion, cc-by-nc-nd)
Funders
Javna Agencija za Raziskovalno Dejavnost RS
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
nanobodies, recombinant protein expression, fusion tags, prokaryotic expression, eukaryotic expression, periplasmic accumulation, cytoplasmic expression, surface display, protein secretion, in silico optimization, immunoreagents
MeSH terms
Animals, Humans, Recombinant Proteins, Gene Expression, Protein Folding, Protein Stability, Single-Domain Antibodies

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