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MAXTIA: A high-throughput platform for rapid functional epitope mapping by kinetic screening of mutant libraries.

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Τίτλος: MAXTIA: A high-throughput platform for rapid functional epitope mapping by kinetic screening of mutant libraries.
Συγγραφείς: Kim K; Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan., Matsunaga R; Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan.; Department of Bioengineering, School of Engineering, The University of Tokyo, Tokyo, Japan., Yokoo T; Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan., Nakakido M; Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan.; Department of Bioengineering, School of Engineering, The University of Tokyo, Tokyo, Japan., Tsumoto K; Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan.; Department of Bioengineering, School of Engineering, The University of Tokyo, Tokyo, Japan.; The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Πηγή: Protein science : a publication of the Protein Society [Protein Sci] 2026 Sep; Vol. 35 (9), pp. e70769.
Τύπος έκδοσης: Journal Article; Research Support, Non-U.S. Gov't
Γλώσσα: English
Στοιχεία περιοδικού: Publisher: Cold Spring Harbor Laboratory Press Country of Publication: United States NLM ID: 9211750 Publication Model: Print Cited Medium: Internet ISSN: 1469-896X (Electronic) Linking ISSN: 09618368 NLM ISO Abbreviation: Protein Sci Subsets: MEDLINE
Imprint Name(s): Publication: 2001- : Woodbury, NY : Cold Spring Harbor Laboratory Press
Original Publication: New York, N.Y. : Cambridge University Press, c1992-
Ιατρικοί όροι (MeSH): Epitope Mapping*/methods , Epitopes*/chemistry , Epitopes*/genetics , Epitopes*/immunology , High-Throughput Screening Assays*/methods , C-Reactive Protein*/chemistry , C-Reactive Protein*/genetics , C-Reactive Protein*/immunology, Kinetics ; Surface Plasmon Resonance ; Models, Molecular ; Mutation
Περίληψη: Accurate identification of epitope residues is essential for developing biopharmaceuticals and understanding the mechanisms of immune recognition. However, experimental approaches for residue-level epitope mapping remain time-consuming and labor-intensive, while accurate computational prediction of protein-protein interfaces remains challenging. Here, we present MAXTIA, a high-throughput kinetic screening platform that integrates cell-free protein synthesis with high-throughput surface plasmon resonance and demonstrate its application to alanine scanning-based functional epitope mapping. This workflow enables the rapid preparation and kinetic characterization of up to 384 protein variants, allowing the identification of functional epitope residues within 3 days while simultaneously providing binding affinity and kinetic parameters (KD, kon, and koff). We applied MAXTIA to map the epitope of the single-domain antibody (VHH) N1 against the pentraxin domain of neuronal pentraxin-2 (NP2 PTX). Alanine substitutions that cause substantial affinity losses clustered within a localized region on the AlphaFold3-predicted NP2 PTX structure, defining a functional epitope site. These residues closely matched the interface observed in the NP2 PTX-VHH N1 crystal structure, validating the accuracy of MAXTIA. Beyond epitope identification, MAXTIA provides a simple and versatile platform for the quantitative analysis of protein-protein interactions, including high-throughput screening of antibody variants for affinity optimization. This approach should accelerate biopharmaceutical development and facilitate mechanistic studies of molecular recognition.
(© 2026 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)
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Grant Information: JPMJAX222I Japan Science and Technology Agency; JP223fa627001 Japan Agency for Medical Research and Development; JP223fa727002 Japan Agency for Medical Research and Development; 22K21343 Japan Society for the Promotion of Science London; JPMXP1122714694 Ministry of Education, Culture, Sports, Science and Technology
Contributed Indexing: Keywords: cell‐free protein synthesis; epitope mapping; high‐throughput experimental system; interaction analysis; kinetic screening; protein engineering
Substance Nomenclature: 0 (Epitopes)
9007-41-4 (C-Reactive Protein)
Entry Date(s): Date Created: 20260817 Date Completed: 20260817 Latest Revision: 20260902
Update Code: 20260903
PubMed Central ID: PMC13479966
DOI: 10.1002/pro.70769
PMID: 42606277
Βάση Δεδομένων: MEDLINE
Περιγραφή
ISSN:1469-896X
DOI:10.1002/pro.70769