Multiple Phagemids Within Single Bacterial Colonies During Construction and Bio-Panning of Single-Domain (VH and VHH) Phage Display Libraries: A Retransformation Strategy for Reliable Clone Screening
5 affiliations
- 1Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
- 2Cancer Research Institute, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
- 3Interdisciplinary Program in Cancer Biology Major, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
- 4Department of Biomedical Science, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
- 5BioMonster Inc., Samsung Cancer Research Building, Seoul National University, Seoul 03080, Republic of Korea
Abstract
Background/Objectives: Phage display relies on the physical linkage between the antibody fragment displayed on a virion (phenotype) and the phagemid encoding it (genotype), allowing antigen-binding clones enriched by bio-panning to be identified by sequencing of individual colonies. In colony-based screening, an additional practical assumption is that each isolated bacterial colony represents a single phagemid sequence. Single-domain antibodies (sdAbs), including the variable domain of the heavy chain of a heavy-chain-only antibody (VHH), are increasingly used as displayed molecules, yet their library construction and bio-panning protocols have been adopted, largely unmodified, from those established for single-chain variable fragment (scFv) and fragment antigen-binding (Fab). We therefore asked whether the underlying assumption of one phagemid per colony holds for sdAb phage display. Methods: Using two alpaca immune libraries—a VHH library and a VH/VHH library (VH, the variable domain of the heavy chain)—we tracked Sanger sequencing chromatograms through library construction and three rounds of bio-panning, and compared classical single-colony screening with a strategy in which pooled phagemid DNA recovered after bio-panning was retransformed into naïve Escherichia coli (E. coli) before screening. Results: Mixed-peak chromatograms were infrequent in the initial libraries (10–20%) but accounted for 80% and 50% of chromatograms in the final bio-panning round of the VHH and VH/VHH libraries. Classical screening—periplasmic enzyme-linked immunosorbent assay (ELISA) followed by Sanger sequencing—failed: only 5.7% and 6.9% of antigen-binding colonies yielded interpretable single-peak sequences. Retransformation restored clonality, raising the proportion of single-peak chromatograms to ~88% overall, and retransformation of individual mixed colonies showed that single colonies harbored multiple distinct phagemids. Conclusions: Colony-level clonality is not strictly preserved in single-domain VH and VHH phage display—more plausibly through multiple phage infection during amplification—and a simple retransformation step restores reliable identification of antigen-binding clones.
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BibTeX
@article{kim2026multiple,
title = {{Multiple Phagemids Within Single Bacterial Colonies During Construction and Bio-Panning of Single-Domain (VH and VHH) Phage Display Libraries: A Retransformation Strategy for Reliable Clone Screening}},
author = {Kyeongeun Kim and Wookyoung Chun and Hyunji Yang and Yonghee Lee and Junho Chung},
journal = {Biomolecules},
year = {2026},
month = oct,
doi = {10.3390/biom16101523},
url = {https://www.mdpi.com/2218-273X/16/10/1523},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Multiple Phagemids Within Single Bacterial Colonies During Construction and Bio-Panning of Single-Domain (VH and VHH) Phage Display Libraries: A Retransformation Strategy for Reliable Clone Screening
AU - Kyeongeun Kim
AU - Wookyoung Chun
AU - Hyunji Yang
AU - Yonghee Lee
AU - Junho Chung
PY - 2026
DA - 2026/10/01
JO - Biomolecules
DO - 10.3390/biom16101523
UR - https://www.mdpi.com/2218-273X/16/10/1523
AB - Background/Objectives: Phage display relies on the physical linkage between the antibody fragment displayed on a virion (phenotype) and the phagemid encoding it (genotype), allowing antigen-binding clones enriched by bio-panning to be identified by sequencing of individual colonies. In colony-based screening, an additional practical assumption is that each isolated bacterial colony represents a single phagemid sequence. Single-domain antibodies (sdAbs), including the variable domain of the heavy chain of a heavy-chain-only antibody (VHH), are increasingly used as displayed molecules, yet their library construction and bio-panning protocols have been adopted, largely unmodified, from those established for single-chain variable fragment (scFv) and fragment antigen-binding (Fab). We therefore asked whether the underlying assumption of one phagemid per colony holds for sdAb phage display. Methods: Using two alpaca immune libraries—a VHH library and a VH/VHH library (VH, the variable domain of the heavy chain)—we tracked Sanger sequencing chromatograms through library construction and three rounds of bio-panning, and compared classical single-colony screening with a strategy in which pooled phagemid DNA recovered after bio-panning was retransformed into naïve Escherichia coli (E. coli) before screening. Results: Mixed-peak chromatograms were infrequent in the initial libraries (10–20%) but accounted for 80% and 50% of chromatograms in the final bio-panning round of the VHH and VH/VHH libraries. Classical screening—periplasmic enzyme-linked immunosorbent assay (ELISA) followed by Sanger sequencing—failed: only 5.7% and 6.9% of antigen-binding colonies yielded interpretable single-peak sequences. Retransformation restored clonality, raising the proportion of single-peak chromatograms to ~88% overall, and retransformation of individual mixed colonies showed that single colonies harbored multiple distinct phagemids. Conclusions: Colony-level clonality is not strictly preserved in single-domain VH and VHH phage display—more plausibly through multiple phage infection during amplification—and a simple retransformation step restores reliable identification of antigen-binding clones.
SN - 2218-273X
ER -