SummaryTo study how the P19 suppressor of gene-silencing protein can be used effectively for the production of therapeutic glycoproteins, the following factors were examined: the genetic elements used for expressing recombinant proteins; the effect of different P19 concentrations; compatibility of P19 with various Nicotiana tabacum cultivars for transgenic expression; the glycan profile of a recombinant therapeutic glycoprotein co-expressed with P19 in an RNAibased glycomodified Nicotiana benthamiana expression host. The coding sequences for the heavy and light chains of trastuzumab were cloned into five plant expression vectors (102-106) containing different 5′ and 3′ UTRs, designated as vector sets 102-106 mAb. The P19 protein of Tomato bushy stunt virus (TBSV) was also cloned into vector 103, which contained the Cauliflower mosaic virus (CaMV) 35S promoter and 5′UTR together with the terminator region of the nopaline synthase gene of Agrobacterium. Transient expression of the antibody vectors resulted in different levels of trastuzumab accumulation, the highest being 105 and 106 mAb at about 1% of TSP. P19 increased the concentration of trastuzumab approximately 15-fold (to about 2.3% of TSP) when co-expressed with 103 mAb but did not affect antibody levels with vectors 102 and 106 mAb. When 103 mAb was expressed together with P19 in different N. tabacum cultivars, all except Little Crittenden showed a marked discolouring of the infiltrated areas of the leaf and decreased antibody expression. Co-expression of P19 also abolished antibody accumulation in crosses between N. tabacum cv. I-64 and Little Crittenden, indicating a dominant mode of inheritance for the observed P19-induced responses.

Expression and purification of recombinant proteins produced in plants is emerging as an affordable alternative to using more costly mammalian bioreactors since plants are capable of producing mammalian proteins at high concentrations. There are two general methods of expressing foreign proteins in plants, namely, transient expression and stable transgenic expression. Both methods have advantages which serve different purposes. Nicotiana benthamiana is primarily used as plant host for transient expression of foreign proteins. This system is capable of producing high yields of antibody in a relatively short period of time (days); however, intensive upstream processing is required as each plant must be infected with Agrobacterium tumefaciens cells by vacuum infiltration. N. tabacum is often used for production of stable transgenic plants through a procedure that requires longer development time (months). Although antibody yields are smaller compared with the transient method, the advantage of using stable transgenic expression is that very little upstream process management is required once homozygous seed lines are developed. In this chapter, we describe the basic methodologies for expressing antibodies in plants using the transient and transgenic systems.

Alcohol dehydrogenase (ADH) activity in plants is generally associated with glycolytic fermentation, which facilitates cell survival during episodes of low-oxygen stress in water-logged roots as well as chronically hypoxic regions surrounding the vascular core. Work with tobacco and potato has implicated ADH activity in additional metabolic roles, including aerobic fermentation, acetaldehyde detoxification and carbon reutilization. Here a combination of approaches has been used to examine tissue-specific patterns of Adh gene expression in order to provide insight into the potential roles of alcohol dehydrogenases, using Petunia hybrida, a solanaceous species with well-characterized genetics. A reporter-gene study, relying on the promoters of Adh1 and Adh2 to drive expression of the gene for a green fluorescent protein derivative, mgfp5, revealed unexpectedly complex patterns of GFP fluorescence in floral tissues, particularly the stigma, style and nectary. Results of GC-MS analysis suggest the association of ADH with production of aromatic compounds in the nectary. Overall the results demonstrate selective recruitment of Adh gene family members in tissues and organs associated with diverse ADH functions.

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To study the agricultural production of biosimilar antibodies, trastuzumab (Herceptin) was expressed in Nicotiana benthamiana using the magnICON viral-based transient expression system. Immunoblot analyses of crude plant extracts revealed that trastuzumab accumulates within plants mostly in the fully assembled tetrameric form. Purification of trastuzumab from N. benthamiana was achieved using a scheme that combined ammonium sulfate precipitation with affinity chromatography. Following purification, the specificity of the plant-produced trastuzumab for the HER2 receptor was compared with Herceptin and confirmed by western immunoblot. Functional assays revealed that plant-produced trastuzumab and Herceptin have similar in vitro antiproliferative effects on breast cancer cells that overexpress HER2. Results confirm that plants may be developed as an alternative to traditional antibody expression systems for the production of therapeutic mAbs.