| RRC ID |
87368
|
| Author |
Agustriana E, Murozono K, Nishioka R, Kawaguchi Y, Kimura M, Kamiya N.
|
| Title |
Chemoenzymatic method for site-selective fluorophore conjugation of a native IgG Fab fragment.
|
| Journal |
J Biosci Bioeng
|
| 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.
|
| Published |
2026-4-8
|
| DOI |
10.1016/j.jbiosc.2026.03.005
|
| PII |
S1389-1723(26)00121-0
|
| PMID |
41956928
|
| IF |
2.366
|
| Resource |
| Human and Animal Cells |
SK-BR-3(RCB2132)
MDA-MB-231/luc(RCB5936) |
