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
Affiliations
Tim Wendlandt
Institute of Biomaterials and Biomolecular Systems, Molecular and Synthetic Plant Virology, University of Stuttgart, Pfaffenwaldring 57, 70569 Stuttgart, Germany
Claudia Koch
Institute of Biomaterials and Biomolecular Systems, Molecular and Synthetic Plant Virology, University of Stuttgart, Pfaffenwaldring 57, 70569 Stuttgart, Germany
Beate Britz
Institute of Biomaterials and Biomolecular Systems, Molecular and Synthetic Plant Virology, University of Stuttgart, Pfaffenwaldring 57, 70569 Stuttgart, Germany
Anke Liedek
Institute of Biomaterials and Biomolecular Systems, Molecular and Synthetic Plant Virology, University of Stuttgart, Pfaffenwaldring 57, 70569 Stuttgart, Germany
Nora Schmidt
Institute of Biomaterials and Biomolecular Systems, Molecular and Synthetic Plant Virology, University of Stuttgart, Pfaffenwaldring 57, 70569 Stuttgart, Germany
Institute of Nano- and Biotechnologies, Aachen University of Applied Sciences, 52428 Jülich, Germany
Fabian J. Eber
Department of Mechanical and Process Engineering, Offenburg University of Applied Sciences, 77652 Offenburg, Germany
Holger Jeske
Institute of Biomaterials and Biomolecular Systems, Molecular and Synthetic Plant Virology, University of Stuttgart, Pfaffenwaldring 57, 70569 Stuttgart, Germany
Christina Wege
Institute of Biomaterials and Biomolecular Systems, Molecular and Synthetic Plant Virology, University of Stuttgart, Pfaffenwaldring 57, 70569 Stuttgart, Germany
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 (TVCVPA) 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 TVCVPA and the wild-type subgroup 3 tobamovirus. TVCVPA 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 TVCVPA 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 TVCVPA-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 TVCVPA-assisted electrochemical biosensing via modularly IgG-assembled sensor enzymes.
