Efficient and Reliable Production of Pharmaceuticals in Alfalfa

In 1986, it was shown that tobacco plants and sunflower calluses could express recombinant human growth hormone as a fusion protein [1]. Since then, a diverse range of plant systems has been used for the production of pharmaceuticals [2, 3]. We have developed a production system based on the leaves of alfalfa (Medicago sativa L.), a choice made originally because of the plant’s many favorable agronomic characteristics. Alfalfa is a perennial plant, so vegetative growth can be maintained for many years. For molecular farming, this characteristic, combined with the ease of clonal propagation through stem cutting, makes alfalfa a robust bioreactor with regard to batch-to-batch reproducibility. Among perennial plants, legume forage crops such as alfalfa have the advantage of fixing atmospheric nitrogen, thus reducing the need for fertilizers. Moreover, as a feed fodder crop, alfalfa has benefited from important research aiming to increase leaf protein content, so that today’s varieties produce as much as 30 mg total protein per gram fresh weight. In addition to these appealing agronomic characteristics, biotechnological research has revealed additional benefits for the production of pharmaceuticals in alfalfa. Expression cassettes have been optimized for protein expression in alfalfa leaves. Methods for transient protein expression have been developed so that it is now possible to use agroinfiltration or the transformation of protoplasts for earlystage demonstration and validation steps. In addition, glycosylation studies have shown that alfalfa is capable of producing recombinant glycoproteins with homogenous (uniform) glycosylation patterns. This chapter provides an overview of the tools that have been developed and optimized specifically for the production of pharmaceuticals in alfalfa, with the emphasis on recent technological breakthroughs. The ability of alfalfa leaves to produce complex recombinant proteins of pharmaceutical interest is discussed and illustrated with recent data obtained in our laboratories. Data are presented concerning the production and characterization of alfalfa-derived C5-1, a diagnostic anti-human 1