Comparative analysis of plant transient expression vectors for targeted N-glycosylation
While plant-based transient expression systems have demonstrated their potency to rapidly express economically feasible quantities of complex human proteins, less is known about their compatibility with posttranslational modification control. Here we investigated three commonly used transient expres...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-12-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2022.1073455/full |
| _version_ | 1811288790412558336 |
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| author | Lukas Eidenberger Florian Eminger Alexandra Castilho Herta Steinkellner |
| author_facet | Lukas Eidenberger Florian Eminger Alexandra Castilho Herta Steinkellner |
| author_sort | Lukas Eidenberger |
| collection | DOAJ |
| description | While plant-based transient expression systems have demonstrated their potency to rapidly express economically feasible quantities of complex human proteins, less is known about their compatibility with posttranslational modification control. Here we investigated three commonly used transient expression vectors, pEAQ, magnICON and pTra for their capability to express a multi-component protein with controlled and modified N-glycosylation. Cetuximab (Cx), a therapeutic IgG1 monoclonal antibody, which carries next to the conserved Fc an additional N-glycosylation site (GS) in the Fab-domain, was used as model. While pEAQ and pTra produce fully assembled Cx at similar levels in N. benthamiana, the yield of magnICON-Cx was twice as high. When expressed in wild type plants, both Cx-GSs exhibited typical plant N-glycans decorated with plant-specific xylose and fucose. Likewise, Cx generated in the glycoengineered ΔXTFT line carried mainly complex N-glycans lacking plant specific residues. Exposure to different engineering settings (encompassing stable lines and transient approaches) towards human galactosylation and sialylation resulted in Cx carrying targeted N-glycans at similar quantities using all three expression vectors. Collectively, our results exhibit the universal application of plant-based glycoengineering, thereby increasing the attractivity of the ambitious expression platform. |
| first_indexed | 2024-04-13T03:43:37Z |
| format | Article |
| id | doaj.art-6d4c6b4407e54eafb7c9e47536b70f81 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-04-13T03:43:37Z |
| publishDate | 2022-12-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-6d4c6b4407e54eafb7c9e47536b70f812022-12-22T03:04:05ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852022-12-011010.3389/fbioe.2022.10734551073455Comparative analysis of plant transient expression vectors for targeted N-glycosylationLukas EidenbergerFlorian EmingerAlexandra CastilhoHerta SteinkellnerWhile plant-based transient expression systems have demonstrated their potency to rapidly express economically feasible quantities of complex human proteins, less is known about their compatibility with posttranslational modification control. Here we investigated three commonly used transient expression vectors, pEAQ, magnICON and pTra for their capability to express a multi-component protein with controlled and modified N-glycosylation. Cetuximab (Cx), a therapeutic IgG1 monoclonal antibody, which carries next to the conserved Fc an additional N-glycosylation site (GS) in the Fab-domain, was used as model. While pEAQ and pTra produce fully assembled Cx at similar levels in N. benthamiana, the yield of magnICON-Cx was twice as high. When expressed in wild type plants, both Cx-GSs exhibited typical plant N-glycans decorated with plant-specific xylose and fucose. Likewise, Cx generated in the glycoengineered ΔXTFT line carried mainly complex N-glycans lacking plant specific residues. Exposure to different engineering settings (encompassing stable lines and transient approaches) towards human galactosylation and sialylation resulted in Cx carrying targeted N-glycans at similar quantities using all three expression vectors. Collectively, our results exhibit the universal application of plant-based glycoengineering, thereby increasing the attractivity of the ambitious expression platform.https://www.frontiersin.org/articles/10.3389/fbioe.2022.1073455/fullNicotiana benthamianatransient expressionN-glycosylationplant biotechnologyglycoengineeringIgG1 |
| spellingShingle | Lukas Eidenberger Florian Eminger Alexandra Castilho Herta Steinkellner Comparative analysis of plant transient expression vectors for targeted N-glycosylation Frontiers in Bioengineering and Biotechnology Nicotiana benthamiana transient expression N-glycosylation plant biotechnology glycoengineering IgG1 |
| title | Comparative analysis of plant transient expression vectors for targeted N-glycosylation |
| title_full | Comparative analysis of plant transient expression vectors for targeted N-glycosylation |
| title_fullStr | Comparative analysis of plant transient expression vectors for targeted N-glycosylation |
| title_full_unstemmed | Comparative analysis of plant transient expression vectors for targeted N-glycosylation |
| title_short | Comparative analysis of plant transient expression vectors for targeted N-glycosylation |
| title_sort | comparative analysis of plant transient expression vectors for targeted n glycosylation |
| topic | Nicotiana benthamiana transient expression N-glycosylation plant biotechnology glycoengineering IgG1 |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2022.1073455/full |
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