Academic Journal
Polyphenol oxidase depletion in Nicotiana benthamiana enhances recombinant protein purification and preserves native protein integrity.
| Τίτλος: | Polyphenol oxidase depletion in Nicotiana benthamiana enhances recombinant protein purification and preserves native protein integrity. |
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| Συγγραφείς: | Zheng K; The Plant Chemetics Laboratory, Department of Biology, University of Oxford, OX1 3RB, Oxford, UK., Kaschani F; Chemical Biology, Faculty of Biology, Universität Duisburg-Essen, ZMB, 45141, Essen, Germany., Watts EC; The Plant Chemetics Laboratory, Department of Biology, University of Oxford, OX1 3RB, Oxford, UK., Kaiser M; Chemical Biology, Faculty of Biology, Universität Duisburg-Essen, ZMB, 45141, Essen, Germany., van der Hoorn RAL; The Plant Chemetics Laboratory, Department of Biology, University of Oxford, OX1 3RB, Oxford, UK. |
| Πηγή: | The New phytologist [New Phytol] 2026 Aug; Vol. 251 (3), pp. 1283-1293. Date of Electronic Publication: 2026 May 24. |
| Τύπος έκδοσης: | Journal Article |
| Γλώσσα: | English |
| Στοιχεία περιοδικού: | Publisher: Wiley on behalf of New Phytologist Trust Country of Publication: England NLM ID: 9882884 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1469-8137 (Electronic) Linking ISSN: 0028646X NLM ISO Abbreviation: New Phytol Subsets: MEDLINE |
| Imprint Name(s): | Publication: Oxford : Wiley on behalf of New Phytologist Trust Original Publication: London, New York [etc.] Academic Press. |
| Ιατρικοί όροι (MeSH): | Nicotiana*/enzymology , Nicotiana*/genetics , Nicotiana*/metabolism , Catechol Oxidase*/metabolism , Recombinant Proteins*/isolation & purification , Recombinant Proteins*/metabolism , Plant Proteins*/metabolism , Plant Proteins*/isolation & purification, Plants, Genetically Modified |
| Περίληψη: | Agroinfiltration of Nicotiana benthamiana is widely used for recombinant protein production in plant science and molecular pharming, but enzymatic browning and native protein crosslinking during extraction may limit protein integrity and purification efficiency. We generated genome-edited N. benthamiana lines lacking two polyphenol oxidases (PPOs) and analyzed protein integrity, enzymatic activity profiles, and recombinant protein purification under non-denaturing extraction conditions. PPO-deficient plants showed reduced browning and native protein crosslinking, preserved endogenous proteins at their predicted molecular weights, displayed increased detectable enzyme activities, and achieved a significantly higher recovery and improved purity of a transiently expressed recombinant protein. These findings identify PPO-mediated oxidation as a major bottleneck during protein extraction and demonstrate that PPO depletion enhances recombinant protein purification while preserving native protein integrity. (© 2026 The Author(s). New Phytologist © 2026 New Phytologist Foundation.) |
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| Grant Information: | 101019324 H2020 European Research Council; BB/Y00969X/1 United Kingdom BB_ Biotechnology and Biological Sciences Research Council; DDT00230 United Kingdom BB_ Biotechnology and Biological Sciences Research Council |
| Contributed Indexing: | Keywords: Nicotiana benthamiana; agroinfiltration; biotechnology; crosslinking; molecular pharming; polyphenol oxidase Local Abstract: [Publisher, Chinese] 在本氏烟草中进行农杆菌瞬时侵染是植物科学和分子医药农业中广泛使用的重组蛋白生产方法，但提取过程中发生的酶促褐变和内源蛋白交联可能会限制蛋白的完整性和纯化效率。 我们构建了敲除两个多酚氧化酶基因的本氏烟草株系，并在非变性提取条件下分析了蛋白质完整性、酶活性谱以及重组蛋白的纯化情况。 PPO缺失突变体表现出褐化和内源蛋白交联程度的降低，内源蛋白在其预测分子量处得到保留，并且检测到更高的酶活性，此外在瞬时表达重组蛋白时实现了显著更高的回收率和更优的纯度。 这些结果表明，PPO介导的氧化是蛋白质提取过程中的一个主要瓶颈，同时表明去除PPO可以在保持内源蛋白完整性的同时，提高重组蛋白的纯化效果。. |
| Substance Nomenclature: | EC 1.10.3.1 (Catechol Oxidase) 0 (Recombinant Proteins) 0 (Plant Proteins) |
| Entry Date(s): | Date Created: 20260525 Date Completed: 20260702 Latest Revision: 20260813 |
| Update Code: | 20260813 |
| PubMed Central ID: | PMC13326509 |
| DOI: | 10.1111/nph.71262 |
| PMID: | 42178657 |
| Βάση Δεδομένων: | MEDLINE |
| ISSN: | 1469-8137 |
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| DOI: | 10.1111/nph.71262 |