High-level production of active human TFPI-2 Kunitz domain in plant

Plants are an attractive production system alternative to cell bioreactor not only because of its lower production costs, but also due to its lack of mammalian pathogens and contaminants, plant capacity to generate appropriate eukaryotic folding and in many cases correct post-translational modifications. In recent years, several recombinant proteins and antibodies have been introduced in the biopharmaceutical market, in particular in cancer therapeutics. Kunitz domain 1 (KD1), a domain of Human Tissue Factor Pathway Inhibitor-2 (TFPI-2), has an outstanding potential in cancer treatment because it is a potent inhibitor of extracellular serine proteinases involved in tumor progression and angiogenesis. We present here the expression and purification of active human KD1 in different Nicotiana species as hosts and its stability during the infection process using a construct derived from a Tobacco mosaic virus (TMV) vector. Our purification protocol allows to recover over 100mg of active human KD1 per batch of 1 kg of plant tissue at about 97% purity. The yields are reproducible, being N. benthamiana the best system where higher levels of KD1 are obtained. Recombinant KD1 was also used to produce a high-sensitivity polyclonal antibody able to detect not only KD1 but also full-length TFPI-2. Finally, we show that this platform is a valuable alternative for the large scale production of KD1.