A rapid novel strategy for screening of antibody phage libraries for production, purification, and functional characterization of amber stop codons containing single-chain antibody fragments
Abstract
Phage display antibody (PDA) libraries, allows the rapid isolation and characterization of high specificity monoclonal antibodies for therapeutic and diagnostic applications. However, selection of positive binding clones from synthetic and semi-synthetic libraries has an inherent bias towards clones containing randomly generated amber stop codons, complicating the identification of high affinity binding antibodies. We screened Tomlinson I and J library against receptor binding domain (RBD) of SARS CoV2, eight clones which showed positive binding in phage ELISA, contained one or more amber stop codons in their single-chain antibody fragment (scFv) gene sequences. The presence of amber stop codons within the antibody sequence causes the premature termination of soluble form of scFv expression in nonsuppressor Escherichia coli strain. In the present study, we have used a novel strategy that allows soluble expression of scFvs having amber stop codon in their gene sequences (without phage PIII protein fusion), in the suppressor strain. This strategy of introduction of Ochre (TAA) codon at the junction of scFv and PIII gene, speeds up the initial screening process which is critical for selecting the right scFvs for further studies. Present strategy leads to the identification of a scFv, B8 that binds specifically with nanomolar affinity toward SARS CoV 2 RBD, which otherwise lost in terms of traditional methodology.
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Significant challenges remain in optimizing the experimental antibody workflow despite reported strategies.
“Although several strategies to optimize the experimental workflow have been reported [3,4], significant challenges remain.”
From Computational and artificial intelligence-based methods for antibody development (2023) · cites this paper as [4]
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Computational and artificial intelligence-based methods for antibody development
Kim, J., McFee, M., Fang, Q., Abdin, O., & Kim, P. M. (2023) · Trends in Pharmacological Sciences · doi:10.1016/j.tips.2022.12.005 · Cited by 178 · see citing works
- Authors
- Perween, R., Ahmed, S., Shrivastava, T., Parray, H. A., Singh, B., Pindari, K. S., Sharma, C., Shukla, S., Sinha, S., Panchal, A. K., & Kumar, R.
- Affiliations
- Translational Health Science and Technology Institute (IN)
- All India Institute of Medical Sciences (IN)
- Corresponding author
- Kumar, Rajesh
- ORCID
- Venue
- Biotechnology Progress
- Publisher
- Wiley
- Published
- 2021-03-08
- Volume
- 37
- Issue
- 3
- Pages
- e3136-e3136
- ISSN
- 1520-6033 (ISSN-L)
- 8756-7938
- OpenAlex
- W3129483641
- Cited by
- 13
- References
- 15
- Citations by year
- 2025: 2 · 2023: 2 · 2022: 5 · 2021: 4
- FWCI
- 1.45
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biotechnology Progress (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Department of Biotechnology, Government of West Bengal (T001)
- 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
- Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
- Keywords
- phage display, amber stop codon, single-chain antibody fragments, scFv, SARS-CoV-2 receptor-binding domain, monoclonal antibody screening, nanomolar affinity
- MeSH terms
- COVID-19, SARS-CoV-2, Antibodies, Monoclonal, Bacteriophages, Humans, Immunoglobulin Variable Region, Protein Binding, RNA, Viral, Codon, Terminator, Peptide Library, Single-Chain Antibodies
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