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bioRxiv · 10.1101/2025.09.28.679031

Polyphenol oxidase mutant Nicotiana benthamiana plants increase yield and purity of recombinant proteins and enable studies of proteins in their native state.

Zheng, K.·van der Hoorn, R. A. L.
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Abstract

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 analysed 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.

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Zheng, K., van der Hoorn, R. A. L.. 2025-09-30. Polyphenol oxidase mutant Nicotiana benthamiana plants increase yield and purity of recombinant proteins and enable studies of proteins in their native state.. https://doi.org/10.1101/2025.09.28.679031

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