Harnessing biomolecular click reactions for modular protein engineering and functionalization

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School of Chemical Engineering | Doctoral thesis (article-based) | Defence date: 2026-03-06
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Date

2026

Department

Biotuotteiden ja biotekniikan laitos
Department of Bioproducts and Biosystems

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Mcode

Degree programme

Language

en

Pages

91 + app. 107

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Aalto University publication series Doctoral Theses, 60/2026

Abstract

Proteins are essential building blocks for advanced biological and bioinspired materials. Their extraordinary structural diversity, modularity, and functional versatility enable the programmable design of intricate materials and architectures. However, fully harnessing the potential of proteins remains challenging. Achieving precise control over protein assembly, constructing multicomponent architectures, and introducing site-specific functionalities are often hindered by the limited availability of covalent protein conjugation tools, which tend to lack versatility and orthogonality. Biomolecular click reactions present promising solutions by expanding the ligation toolkit with genetically encodable and efficient reactions, thus facilitating reliable and modular protein assembly and functionalization. In this thesis, I systematically explored the applications of Catcher/Tag-mediated biomolecular click reactions as a modular platform for protein assembly and functionalization, employing spider-silk-like proteins as primary model building blocks. I engineered a novel minimal Catcher/Tag pair, termed SilkCatcher/SilkTag (SilkC/SilkT), derived from the CnaB1 domain of the cell surface protein lp2578 from Lactobacillus plantarum (L. plantarum). The SilkC/SilkT system demonstrated efficient formation of covalent isopeptide bonds over a broad range of mild conditions and exhibited a distinct pH-activated reaction. Its reaction profile was comparable to the well-established SpyCatcher/SpyTag (SpyC/SpyT) system, while its ligation specificity remained strictly orthogonal to the SpyC/SpyT pair, thereby expanding the toolkit available for orthogonal protein ligations. Fusion of Catcher domains to aggregation-prone proteins, including spider-silk-like proteins and β-glucosidase, significantly enhanced their soluble expression in Escherichia coli (E. coli). The improved solubility of spider-silk-like proteins enabled the production of artificial spider silk fibers, exhibiting outstanding extensibility and toughness, via a fully aqueous wet-spinning system. The intrinsic structural affinity and covalent reactivity between Catcher domains and Tag peptides were further leveraged for multiple functional applications, such as site-specific fiber functionalization, enzyme immobilization, protein purification, as well as controlled polyphosphorylation of spider-silk-like proteins. Additionally, the compatible reaction conditions and orthogonality of the SilkC/SilkT and SpyC/SpyT pairs facilitated multiple-fragment protein conjugation, allowing the construction of native-sized and ultrahigh-molecular-weight spider-silk-like proteins. In summary, this thesis demonstrates that the Catcher/Tag-mediated biomolecular click reactions provide a versatile and modular platform for protein engineering. Their efficient covalent ligation and strict orthogonality enable controlled protein assembly, targeted immobilization, and precise functional modification. These studies highlight the broad potential of biomolecular click reactions for advanced protein engineering and their use for the development of functional biomaterials.

Description

Supervising professor

Aranko, Sesilja, Assist. Prof., Aalto University, Department of Bioproducts and Biosystems, Finland

Thesis advisor

Aranko, Sesilja, Assist. Prof., Aalto University, Department of Bioproducts and Biosystems, Finland

Keywords

biomolecular click reaction, catcher/tag, spider silk, modular protein assembly, functionalization

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Parts

  • [Publication 1]: Fan, Ruxia; Hakanpää, Johanna; Elfving, Karoliina; Taberman, Helena; Linder, Markus B.; Aranko, A. Sesilja. 2023. Biomolecular Click Reactions Using a Minimal pH‐Activated Catcher/Tag Pair for Producing Native‐Sized Spider‐Silk Proteins. Angewandte Chemie International Edition, 62, 11, e202216371.
    Full text in Acris/Aaltodoc: https://urn.fi/URN:NBN:fi:aalto-202305243386
    DOI: 10.1002/anie.202216371 View at publisher
  • [Publication 2]: Fan, Ruxia; Wan, Xing; Khanum, Safoorah; Jokio, Juhani P.; Möttönen, Nea B.; Mäkelä, Miia R.; Aranko, A. Sesilja. 2026. Catcher Domains as Multifunctional Fusion Modules for Soluble Expression, Covalent Coupling, and Spatial Organization of Recombinant Proteins (Submitted)
  • [Publication 3]: Fan, Ruxia; Knuuttila, Katarina; Schmuck, Benjamin; Greco, Gabriele; Rising, Anna; Linder, Markus B.; Aranko, A. Sesilja. 2025. Sustainable Spinning of Artificial Spider Silk Fibers with Excellent Toughness and Inherent Potential for Functionalization. Advanced Functional Materials, 35, 15, 2410415.
    Full text in Acris/Aaltodoc: https://urn.fi/URN:NBN:fi:aalto-202505143632
    DOI: 10.1002/adfm.202410415 View at publisher
  • [Publication 4]: Möttönen, Nea B.; Fan, Ruxia; Aspholm-Tsironi, Stefania; Keskitalo, Salla; Tuhkala, Antti; Varjosalo, Markku; Aranko, A. Sesilja. 2025. Induced proximity approach enables the recombinant production of polyphosphorylated silk proteins with improved adhesiveness. Biomacromolecules, 26, 12, 8594-8605.
    Full text in Acris/Aaltodoc: https://urn.fi/URN:NBN:fi:aalto-202601141195
    DOI: 10.1021/acs.biomac.5c01431 View at publisher

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https://urn.fi/URN:ISBN:978-952-64-3035-5

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[diss] Kemian tekniikan korkeakoulu / CHEM