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

Agroinfiltration of Nicotiana benthamiana is frequently used to produce recombinant proteins, both for plant science and for molecular pharming. Here, we introduce two genome-edited lines of N. benthamiana lacking two polyphenol oxidases (PPOs). These double ppo knockout lines grow slightly faster than wild-type and show similar levels of transient GFP expression. However, leaf extracts produced in native buffers stay greener and show much less native crosslinking of Rubisco and other proteins, demonstrating that PPO depletion reduces enzymatic browning and protein crosslinking in leaf extracts. Transient PPO1 expression in the ppo mutant restores browning and crosslinking in leaf extracts. These ppo mutants offer tremendous opportunities to increase yield and purity of recombinant proteins and study protein complexes, as illustrated with a nearly 4-fold increase in purification yield and a substantial improvement of protein purity upon purification of transiently expressed His-tagged tomato immune protease P69B from total leaf extracts.