The plant-specific glycosylation including the 1, 2-xylose and a core 1, 3-fucose takes place in the Golgi apparatus. plants, faithfully inherited, and expressed in later generations. Furthermore, plant-derived recombinant proteins may be safer than those from or mammalian cells because the risk of contamination with human pathogens, Rabbit polyclonal to NPSR1 which is always a concern when using mammalian cells as a bioreactor, can be well circumvented by plant-based production systems (Thie et al., 2008; Ni and Chen, 2009; Merlin et al., 2014). Because of these properties, various bioactive pharmaceutical proteins have been produced in plants since first expression of a human growth hormone in transgenic tobacco and sunflower callus tissue (Barta et al., 1986), and expression of antibodies, vaccines, hormones, growth factors, and cytokines (De Muynck et Oleanolic acid hemiphthalate disodium salt al., 2010; Desai et al., 2010; Xu et al., 2011; Huang and McDonald, 2012). Monoclonal antibodies (mAbs) are protein complexes containing four subunits with two identical light chains (LC) and two identical heavy chains (HC). MAbs are important in biological research, clinical diagnosis, and recently immunotherapy for various diseases and cancer (De Muynck et al., 2010). Unlike other single chain recombinant proteins, the production of mAbs requires simultaneous expression of two genes coding for both LC and HC, and the correct folding of four polypeptides linked by disulphide bonds. The discovery that plant can efficiently express and correctly assemble functional antibodies (Hiatt et al., 1989) have made plants an Oleanolic acid hemiphthalate disodium salt alternative antibody production system, and since then, many recombinant antibodies have been produced in various plants, including moss (Decker and Reski, 2008), algae (Franklin and Oleanolic acid hemiphthalate disodium salt Mayfield, 2005), and higher plants (Stoger et al., 2005; De Muynck et al., 2010; Xu et al., 2011, 2012; Schillberg et al., 2013). Previously, the LC and HC genes of a mAb were expressed in two different expression cassettes on one T-DNA region of a vector, or expressed separately in individual vectors which were co-transformed to the same plant, or expressed in different transgenic plants which were cross-fertilized to produce the functional antibody (De Muynck et al., 2010; Ko, 2014). Because the two genes are expressed separately, it is difficult to control their relative expression level even though identical regulatory elements are used. In fact, most of the previous reports have produced unbalanced LC and HC in both transgenic plants and mammalian cells (Voss et al., 1995; Law et al., 2006; De Muynck et al., 2010; Chng et al., 2015). A varied LC:HC ratio is usually unfavorable for the folding of functional mAbs, and affects both the level and quality of mAbs (Schlatter et al., 2005; Law et al., 2006; Lee et al., 2009; Ho et al., 2013b). The use of internal ribosome entry site (IRES) to translate two polypeptides (LC and HC) from one mRNA also results in an unbalanced expression because of the lower efficiency of the IRES directed downstream gene expression by a cap-independent translation mechanism (Hennecke et al., 2001; Ho et al., 2012, 2013a,b). In contrast, the use of 2A peptide from the Aphthovirus (FMDV) for high level mAb expression has been reported in both the human embryonic kidney 293 and the Chinese hamster ovary (CHO) cells (Fang et al., 2005; Chng et al., 2015), but this strategy for mAb expression in transgenic plant system has not been reported so far. Bevacizumab is a humanized mAb that targets to the vascular endothelial growth factor (VEGF) antigen (Presta et al., 1997; Ferrara et al., 2005), which is widely over expressed in a variety of human solid tumors and plays a key role in tumor angiogenesis (Ellis and Hicklin, 2008; Goel and Mercurio, 2013; Domigan et al., 2015). Bevacizumab neutralizes VEGFs, prevents their interactions with VEGFR-1 and VEGFR-2 receptors, and thus blocks the downstream signal transductions for tumor angiogenesis (Wang et al., 2004). Bevacizumab is derived from the murine VEGF mAb A4.6.1. It has 93% human and 7% murine sequence, and has similar biochemical and pharmacologic properties to the original murine mAb. It neutralizes all isoforms of human VEGF (hVEGF) with high affinity and inhibits VEGF-induced proliferation of endothelial cells and tumor angiogenesis, but with reduced immunogenicity and longer circular half-life as compared to the murine antibody (Gerber and Ferrara, 2005). It has been widely applied in clinical practice of metastatic colorectal cancer, glioblastoma, non-small-cell lung cancer, metastatic kidney.