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Opdensteinen, P.* ; Sperl, L.E. ; Mohamadi, M. ; Kündgen-Redding, N.* ; Hagn, F. ; Buyel, J.F.*

The transient expression of recombinant proteins in plant cell packs facilitates stable isotope labeling for NMR spectroscopy.

Plant Biotechnol. J. 20, 1928-1939 (2022)

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Nuclear magnetic resonance (NMR) spectroscopy can be used to determine the structure, dynamics and interactions of proteins. However, protein NMR requires stable isotope labeling for signal detection. The cells used for the production of recombinant proteins must therefore be grown in medium containing isotopically labeled substrates. Stable isotope labeling is well established in Escherichia coli, but bacteria are only suitable for the production of simple proteins without post-translational modifications. More complex proteins require eukaryotic production hosts, but their growth can be impaired by labeled media, thus reducing product yields and increasing costs. To address this limitation, we used media supplemented with isotope-labeled substrates to cultivate the tobacco-derived cell line BY-2, which was then cast into plant cell packs (PCPs) for the transient expression of a labeled version of the model protein GB1. Mass spectrometry confirmed the feasibility of isotope labeling with ¹⁵ N and ² H using this approach. The resulting NMR spectrum featured a signal dispersion comparable to recombinant GB1 produced in E. coli. PCPs therefore offer a rapid and cost-efficient alternative for the production of isotope-labeled proteins for NMR analysis, especially suitable for complex proteins that cannot be produced in microbial systems.

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Publication type Article: Journal article

Document type Scientific Article

Corresponding Author

Keywords Alternative Expression Host ; Defined Cultivation Media ; Isotope Labeling ; Plant Cell Culture ; Structural Analysis

ISSN (print) / ISBN 1467-7644

e-ISSN 1467-7652

Journal Plant Biotechnology Journal

Quellenangaben Volume: 20, Issue: 10, Pages: 1928-1939

Publisher Wiley

Non-patent literature Publications

Reviewing status Peer reviewed

Institute(s) Institute of Structural Biology (STB)