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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]
Copyright of Journal of Bioscience & Bioengineering is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
Database: Engineering Source
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Header DbId: egs
DbLabel: Engineering Source
An: 193621617
AccessLevel: 6
PubType: Academic Journal
PubTypeId: academicJournal
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Items – Name: Title
  Label: Title
  Group: Ti
  Data: Chemoenzymatic method for site-selective fluorophore conjugation of a native IgG Fab fragment.
– Name: Author
  Label: Authors
  Group: Au
  Data: <searchLink fieldCode="AR" term="%22Agustriana%2C+Eva%22">Agustriana, Eva</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Murozono%2C+Koki%22">Murozono, Koki</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Nishioka%2C+Riko%22">Nishioka, Riko</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Kawaguchi%2C+Yoshirou%22">Kawaguchi, Yoshirou</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Kimura%2C+Michio%22">Kimura, Michio</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Kamiya%2C+Noriho%22">Kamiya, Noriho</searchLink><relatesTo>1,2</relatesTo> (AUTHOR)<i> kamiya.noriho.367@m.kyushu-u.ac.jp</i>
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  Data: <searchLink fieldCode="JN" term="%22Journal+of+Bioscience+%26+Bioengineering%22">Journal of Bioscience & Bioengineering</searchLink>. Jul2026, Vol. 142 Issue 1, p38-44. 7p.
– Name: Subject
  Label: Subjects
  Group: Su
  Data: <searchLink fieldCode="DE" term="%22Click+chemistry%22">Click chemistry</searchLink><br /><searchLink fieldCode="DE" term="%22Bioconjugates%22">Bioconjugates</searchLink><br /><searchLink fieldCode="DE" term="%22Trastuzumab%22">Trastuzumab</searchLink><br /><searchLink fieldCode="DE" term="%22Fluorescent+probes%22">Fluorescent probes</searchLink><br /><searchLink fieldCode="DE" term="%22Transglutaminases%22">Transglutaminases</searchLink><br /><searchLink fieldCode="DE" term="%22Immunoglobulins%22">Immunoglobulins</searchLink>
– Name: Abstract
  Label: Abstract
  Group: Ab
  Data: 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]
– Name: AbstractSuppliedCopyright
  Label:
  Group: Ab
  Data: <i>Copyright of Journal of Bioscience & Bioengineering is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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RecordInfo BibRecord:
  BibEntity:
    Identifiers:
      – Type: doi
        Value: 10.1016/j.jbiosc.2026.03.005
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      – Code: eng
        Text: English
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      Pagination:
        PageCount: 7
        StartPage: 38
    Subjects:
      – SubjectFull: Click chemistry
        Type: general
      – SubjectFull: Bioconjugates
        Type: general
      – SubjectFull: Trastuzumab
        Type: general
      – SubjectFull: Fluorescent probes
        Type: general
      – SubjectFull: Transglutaminases
        Type: general
      – SubjectFull: Immunoglobulins
        Type: general
    Titles:
      – TitleFull: Chemoenzymatic method for site-selective fluorophore conjugation of a native IgG Fab fragment.
        Type: main
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            NameFull: Agustriana, Eva
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            NameFull: Murozono, Koki
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            NameFull: Nishioka, Riko
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            NameFull: Kawaguchi, Yoshirou
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            NameFull: Kimura, Michio
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            – D: 01
              M: 07
              Text: Jul2026
              Type: published
              Y: 2026
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              Value: 142
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