Rapid design and production of Alpha-1 antitrypsin fusion proteins in a plant-based cell-free expression system.

Alpha-1 antitrypsin deficiency (AATD) is a rare disease, causing significant lung damage, that is currently treated with FDA approved replacement therapy comprised of weekly infusions of costly plasma-derived AAT. Fusing AAT with a crystallizable fragment (Fc) region could potentially extend the plasma half-life, reducing dose frequency. However, computational structural simulations predict steric hindrance of the Fc domain with the linker that effects the binding of AAT-Fc to neutrophil elastase and require functional experimental evaluation. Using the Nicotiana tabacum BY-2 cell derived "Almost Living Cell Free Expression" (ALiCE®) platform, we rapidly produced AAT and AAT-Fc variants and tested their anti-elastase functionality. AAT and AAT-Fc titers were 0.92 mg/mL and 0.57 mg/mL in the ALiCE® reaction lysate after 48-h reactions respectively, AAT showing anti-elastase activity in a band-shift assay. In contrast, AAT-Fc variants lacked band-shift assay activity and had low anti-elastase activity based on residual-elastase assays, the data being in agreement with computational structural prediction. This study demonstrates ALiCE® as an efficient platform for rapid production and analysis of the functional activity of complex glycoproteins.