Vector minimization and infiltration optimization for enhanced protein expression in Nicotiana benthamiana.

key messageVector minimization, combined with optimized infiltration buffer conditions, enhances recombinant protein expression in Nicotiana benthamiana, offering practical improvements in plant-based biotechnology. Nicotiana benthamiana is a widely utilized host for transient expression of recombinant proteins, offering a rapid and flexible system for biotechnology applications. However, achieving high-protein output remains a significant challenge. In this study, we explored how vector minimization and infiltration buffer optimization influence recombinant protein expression. We systematically reduced the size of the commonly used pBI121 vector by eliminating non-essential sequences, generating pBI121Δlac and modified pBI121 (mpBI121) constructs. Using ꞵ-glucuronidase (GUS) and acid sphingomyelinase (ASM) as model proteins, we found that vector minimization significantly enhanced both mRNA and protein expression. The mpBI121 backbone improved GUS and ASM mRNA expression levels by 2.02-fold and 2.05-fold, respectively, versus pBI121. Correspondingly, protein levels increased by 1.76-fold and 1.75-fold. Optimization of infiltration buffer-together with co-expression of the silencing suppressor p19, along with acetosyringone, and ascorbic acid-further improved protein output, producing the highest expression of both proteins. These findings demonstrate that both vector minimization and optimized agroinfiltration conditions enhance recombinant protein expression in plant systems, advancing plant-based biotechnology platforms.