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Chemoenzymatic method for site-selective fluorophore conjugation of a native IgG Fab fragment.

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Title: Chemoenzymatic method for site-selective fluorophore conjugation of a native IgG Fab fragment.
Authors: Agustriana, Eva1 (AUTHOR), Murozono, Koki1 (AUTHOR), Nishioka, Riko1 (AUTHOR), Kawaguchi, Yoshirou1 (AUTHOR), Kimura, Michio1 (AUTHOR), Kamiya, Noriho1,2 (AUTHOR) kamiya.noriho.367@m.kyushu-u.ac.jp
Source: Journal of Bioscience & Bioengineering. Jul2026, Vol. 142 Issue 1, p38-44. 7p.
Subjects: Click chemistry, Bioconjugates, Trastuzumab, Fluorescent probes, Transglutaminases, Immunoglobulins
Abstract: Antibody-derived bioconjugates have emerged as practical biomolecules for applications in diagnostics and therapeutics. A key challenge is achieving site-selective bioconjugation to preserve the native function of the labeled biomolecule. Methods that target native amino acid residues can broaden the applicability of bioconjugates; however, most existing methods rely on genetically engineered antibodies to ensure site-specific labeling. In this report, a chemoenzymatic strategy for modifying a native Fab fragment derived from trastuzumab was investigated using the EzMTG-pG fusion protein, which consists of a microbial transglutaminase variant and protein G. To mitigate the hydrophobic nature of the widely used dibenzocyclooctyne (DBCO) moiety for click chemistry, a new DBCO-containing glutamine donor peptide (DBCO-PEG4-LLQG) was designed. This substrate peptide enabled Lys65-selective conjugation of the Fab via EzMTG-pG catalysis, achieving a 92% modification rate within 4 h. The subsequent click reaction between the Fab-DBCO conjugate and an azide-bearing fluorescent small molecule probe generated a Fab-fluorophore conjugate that retained cell-specific binding to a target cell line. These results demonstrate that the chemoenzymatic pathway, using EzMTG-pG catalysis combined with a click reaction, provides a versatile approach for generating Fab-based bioconjugates with potential applications in diagnostics and therapeutics. • Site-selective enzymatic Fab labeling with a strained alkyne (DBCO) was achieved. • Click reaction of Fab-DBCO with a fluorophore results in a quantitative conversion. • The Fab conjugate retained antigen-binding ability and fluorophore functionality. [ABSTRACT FROM AUTHOR]
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Database: Engineering Source
Description
Abstract:Antibody-derived bioconjugates have emerged as practical biomolecules for applications in diagnostics and therapeutics. A key challenge is achieving site-selective bioconjugation to preserve the native function of the labeled biomolecule. Methods that target native amino acid residues can broaden the applicability of bioconjugates; however, most existing methods rely on genetically engineered antibodies to ensure site-specific labeling. In this report, a chemoenzymatic strategy for modifying a native Fab fragment derived from trastuzumab was investigated using the EzMTG-pG fusion protein, which consists of a microbial transglutaminase variant and protein G. To mitigate the hydrophobic nature of the widely used dibenzocyclooctyne (DBCO) moiety for click chemistry, a new DBCO-containing glutamine donor peptide (DBCO-PEG4-LLQG) was designed. This substrate peptide enabled Lys65-selective conjugation of the Fab via EzMTG-pG catalysis, achieving a 92% modification rate within 4 h. The subsequent click reaction between the Fab-DBCO conjugate and an azide-bearing fluorescent small molecule probe generated a Fab-fluorophore conjugate that retained cell-specific binding to a target cell line. These results demonstrate that the chemoenzymatic pathway, using EzMTG-pG catalysis combined with a click reaction, provides a versatile approach for generating Fab-based bioconjugates with potential applications in diagnostics and therapeutics. • Site-selective enzymatic Fab labeling with a strained alkyne (DBCO) was achieved. • Click reaction of Fab-DBCO with a fluorophore results in a quantitative conversion. • The Fab conjugate retained antigen-binding ability and fluorophore functionality. [ABSTRACT FROM AUTHOR]
ISSN:13891723
DOI:10.1016/j.jbiosc.2026.03.005