Facile Purification and Use of Tobamoviral Nanocarriers for Antibody-Mediated Display of a Two-Enzyme System

Tim Wendlandt; Claudia Koch; Beate Britz; Anke Liedek; Nora Schmidt; Stefan Werner; Yuri Gleba; Farnoosh Vahidpour; Melanie Welden; Arshak Poghossian; Michael J Schöning; Fabian J Eber; Holger Jeske; Christina Wege

doi:10.3390/v15091951

Facile Purification and Use of Tobamoviral Nanocarriers for Antibody-Mediated Display of a Two-Enzyme System

Viruses. 2023 Sep 19;15(9):1951. doi: 10.3390/v15091951.

Authors

Affiliations

¹ Institute of Biomaterials and Biomolecular Systems, Molecular and Synthetic Plant Virology, University of Stuttgart, Pfaffenwaldring 57, 70569 Stuttgart, Germany.
² Nambawan Biotech GmbH/Now at Icon Genetics GmbH, Weinbergweg 22, 06120 Halle, Germany.
³ Nomad Bioscience GmbH, Weinbergweg 22, 06120 Halle, Germany.
⁴ Institute of Nano- and Biotechnologies, Aachen University of Applied Sciences, 52428 Jülich, Germany.
⁵ MicroNanoBio, Liebigstrasse 4, 40479 Düsseldorf, Germany.
⁶ Institute of Biological Information Processing (IBI-3), Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
⁷ Department of Mechanical and Process Engineering, Offenburg University of Applied Sciences, 77652 Offenburg, Germany.

Abstract

Immunosorbent turnip vein clearing virus (TVCV) particles displaying the IgG-binding domains D and E of Staphylococcus aureus protein A (PA) on every coat protein (CP) subunit (TVCV_PA) were purified from plants via optimized and new protocols. The latter used polyethylene glycol (PEG) raw precipitates, from which virions were selectively re-solubilized in reverse PEG concentration gradients. This procedure improved the integrity of both TVCV_PA and the wild-type subgroup 3 tobamovirus. TVCV_PA could be loaded with more than 500 IgGs per virion, which mediated the immunocapture of fluorescent dyes, GFP, and active enzymes. Bi-enzyme ensembles of cooperating glucose oxidase and horseradish peroxidase were tethered together on the TVCV_PA carriers via a single antibody type, with one enzyme conjugated chemically to its Fc region, and the other one bound as a target, yielding synthetic multi-enzyme complexes. In microtiter plates, the TVCV_PA-displayed sugar-sensing system possessed a considerably increased reusability upon repeated testing, compared to the IgG-bound enzyme pair in the absence of the virus. A high coverage of the viral adapters was also achieved on Ta₂O₅ sensor chip surfaces coated with a polyelectrolyte interlayer, as a prerequisite for durable TVCV_PA-assisted electrochemical biosensing via modularly IgG-assembled sensor enzymes.

Keywords: antibody; biosensor; enzyme cascade; enzyme nanocarrier; glucose oxidase (GOx); horseradish peroxidase (HRP); protein A; tobacco mosaic virus (TMV); tobamovirus; turnip vein clearing virus (TVCV).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Fluorescent Dyes*
Immunoglobulin G
Polyelectrolytes
Polyethylene Glycols*

Substances

Fluorescent Dyes
Polyelectrolytes
Polyethylene Glycols
Immunoglobulin G

Grants and funding

Part of this research was funded by the Deutsche Forschungsgemeinschaft (DFG: German Research Foundation), grant number 446507449, and part of the work via a donation by Nomad Bioscience GmbH, Halle, Germany. The article processing charge was funded by the German Research Foundation (DFG) grant “Open Access Publication Funding/2023–2024/University of Stuttgart” (512689491) with subsidiary funding by the University of Stuttgart.