{"title":"Proximity-based site-specific labeling of a native IgG Fab fragment by a fusion microbial transglutaminase-protein G variant","authors":"Koki Murozono , Riko Nishioka , Yoshirou Kawaguchi , Michio Kimura , Noriho Kamiya","doi":"10.1016/j.nbt.2025.08.004","DOIUrl":null,"url":null,"abstract":"<div><div>The fragment antigen-binding (Fab) fragment of IgG has been studied widely as a delivery vehicle for tumor-targeting drugs and dyes due to its high specificity and enhanced tumor penetration, which is attributed to its small size. Functionalizing Fab with chemical entities requires site-specific modification to preserve the binding ninity and ensure product homogeneity. In this study, we report a tag-free, site-specific labeling approach targeting a Lys residue in Fab using the recently developed engineered zymogen of microbial transglutaminase fused with an antibody-binding protein G. Fab of trastuzumab, prepared via papain digestion, was selectively modified at Lys 65 in the heavy chain with a glutamine-donor fluorescent substrate, achieving a high labeling efficiency (∼96 %). Bio-layer interferometry experiments confirmed that the modified Fab retained antigen-binding affinity (<em>K</em><sub>D</sub> = 5.71 ± 3.89 nM) comparable to its native counterpart (4.72 ± 3.19 nM). Confocal microscopy analysis demonstrated selective binding of the fluorescent-modified Fab to human epidermal growth factor receptor type2 (HER2)-positive SK-BR-3 cells, with negligible binding to HER2-negative MDA-MB-231 cells. The proposed strategy enables site-specific Fab modification without genetic engineering, offering a streamlined approach to producing homogeneous Fab conjugates for diagnostic imaging and therapeutic antibody engineering applications.</div></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"90 ","pages":"Pages 56-64"},"PeriodicalIF":4.9000,"publicationDate":"2025-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"New biotechnology","FirstCategoryId":"5","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1871678425000810","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
The fragment antigen-binding (Fab) fragment of IgG has been studied widely as a delivery vehicle for tumor-targeting drugs and dyes due to its high specificity and enhanced tumor penetration, which is attributed to its small size. Functionalizing Fab with chemical entities requires site-specific modification to preserve the binding ninity and ensure product homogeneity. In this study, we report a tag-free, site-specific labeling approach targeting a Lys residue in Fab using the recently developed engineered zymogen of microbial transglutaminase fused with an antibody-binding protein G. Fab of trastuzumab, prepared via papain digestion, was selectively modified at Lys 65 in the heavy chain with a glutamine-donor fluorescent substrate, achieving a high labeling efficiency (∼96 %). Bio-layer interferometry experiments confirmed that the modified Fab retained antigen-binding affinity (KD = 5.71 ± 3.89 nM) comparable to its native counterpart (4.72 ± 3.19 nM). Confocal microscopy analysis demonstrated selective binding of the fluorescent-modified Fab to human epidermal growth factor receptor type2 (HER2)-positive SK-BR-3 cells, with negligible binding to HER2-negative MDA-MB-231 cells. The proposed strategy enables site-specific Fab modification without genetic engineering, offering a streamlined approach to producing homogeneous Fab conjugates for diagnostic imaging and therapeutic antibody engineering applications.
期刊介绍:
New Biotechnology is the official journal of the European Federation of Biotechnology (EFB) and is published bimonthly. It covers both the science of biotechnology and its surrounding political, business and financial milieu. The journal publishes peer-reviewed basic research papers, authoritative reviews, feature articles and opinions in all areas of biotechnology. It reflects the full diversity of current biotechnology science, particularly those advances in research and practice that open opportunities for exploitation of knowledge, commercially or otherwise, together with news, discussion and comment on broader issues of general interest and concern. The outlook is fully international.
The scope of the journal includes the research, industrial and commercial aspects of biotechnology, in areas such as: Healthcare and Pharmaceuticals; Food and Agriculture; Biofuels; Genetic Engineering and Molecular Biology; Genomics and Synthetic Biology; Nanotechnology; Environment and Biodiversity; Biocatalysis; Bioremediation; Process engineering.