== SDS-PAGE analysis to compare the purity of GA733P-FcK with GA733M-Fc after optimization of ammonium sulfate concentration (50%). Black arrowhead Hhex indicates a 65 kDa GA733-Fc protein music group. == Binding Activity of Purified GA733P-FcK to Anti-GA733 mAb == Kinetic analysis of binding activity of GSK-269984A purified GA733P-FcK to anti-GA733 mAb using SPR showed that GA733P-FcK had almost three times higher Ru value than parental GA733M-FcK. fused to the Fc fragment of human IgG tagged with endoplasmic reticulum retention signal KDEL (GA733P-FcK) protein significantly increased 1 . 8-fold. SDS-PAGE analysis showed that the purity of GA733P-FcK protein music group appeared to be just like that of an equal dose of mammalian-derived GA733-Fc (GA733M-Fc). The binding activity of purified GA733P-FcK to anti-GA733 mAb was as effective as the native GA733M-Fc. Thus, the purification process was effectively optimized to get obtaining a large yield of plant-derived antigenic protein with good quality. In conclusion, the purification recovery price of large quantities of recombinant protein from plant expression systems can be enhanced via optimization of ammonium sulfate concentration during downstream processes, thereby offering a promising answer for production of recombinant GA733-Fc protein in plants. Keywords: ammonium sulfate, colorectal cancer, GA733-Fc, recombinant vaccine, transgenic herb == Intro == Mammalian, yeast, and insect cell cultures are used to produce recombinant subunit vaccines owing to their ability to express proteins in a manner just like that observed in the native organism (Ko, 2014). However , expensive culture media and purification methods required for recovering recombinant proteins expressed in the cells of those organisms increase the cost of recombinant subunit vaccine production. In addition , most subunit vaccines produced in these systems are heat sensitive and require parenteral delivery, which restricts the use of recombinant subunit vaccines in many developing countries where wellness systems are not well equipped (Rigano and Walmsley, 2005). For use in herb molecular biofarming, the cigarette plant offers several advantages over other plants, such as highly effective transformation and regeneration, relatively short period to get biomass production, and easy homogenization processing to get protein purification due to soft leaf cells (Jamal et al., 2009, 2012; Song et al., 2015). The tumor-associated antigen GA733, a glycoprotein that is highly expressed on the cell-surface of colorectal carcinomas, offers previously been fused GSK-269984A with all the human immunoglobulin Fc fragment carrying the ER retention sequence, to produce the recombinant antigen-antibody complex GA733-FcK in a plant expression system (Lu et al., 2012; Park et al., 2015a, b). Moreover, expression of the recombinant protein GA733-FcK has been successful in a herb system (Lim et al., 2015). Optimization of the protein purification process is essential to get the successful production from the tagged recombinant therapeutic protein in the total soluble protein (TSP) recovery step during the downstream process (Geyer et al., 2005; Aspelund and Glatz, 2010; Kim et al., 2015). To purify the tagged recombinant proteins, the TSPs must be extracted, separated, and eventually isolated, from the plant biomass debris (Desai et al., 2002). Generally, 35% ammonium sulfate is often applied for precipitation of TSPs from herb extracts (Lim et al., 2015). However , the ammonium sulfate concentration for the precipitation of TSPs harboring GA733-FcK has not been optimized. In this study, a recombinant colorectal cancer vaccine candidate fusion protein GA733-FcK expressed in transgenicNicotiana tabacumplants was purified by protein-G affinity chromatography. Thus, we optimized ammonium sulfate TSP precipitation conditions to increase the recovery price of GA733-FcK in transgenic plant leaves. == Components and Methods == == Plant Material == Seedlings of transgenic tobacco (N. tabacum) plants expressing the recombinant protein GA733-FcK (Lu et al., 2012) were transplanted into a pot that contain soil and grown in a greenhouse (Figure1A). == PHYSIQUE 1 . == GSK-269984A Growth of transgenic plants expressing GA733-FcK (A) in a greenhouse and immunoblot analysis from the plant-derived GA733-FcK (GA733P-FcK) and mammalian-derived GA733-Fc (GA733M-Fc) (B). Plant leaf samples were homogenized in 1 PBS, separated by 10% SDS-PAGE, and transferred onto nitrocellulose membrane. Lane 1, positive control; GA733M-Fc (68 kDa); lane 2, non-transgenic herb (NT); and lane three or more, GA733P-FcK. The bands were detected by anti-human Fc IgG conjugated to HRP. == Removal of Chloroplasts == For purification of the plant-derived recombinant protein GA733-FcK (GA733P-FcK), tobacco herb leaves were homogenized in an HR2094 blender (Philips, Seoul, Korea) using extraction buffer (37. 5 mM Tris-HCl, pH 7. 5; 50 mM NaCl; 15 mM EDTA; 75 mM sodium citrate; and 0. 2% sodium thiosulfate) (Figure2). After centrifugation at 9, 000 gfor 30 min at 4C, the supernatant was filtered through a Miracloth (Biosciences, La Jolla, CA, USA), and pH of the filtered solution reduced to 5. 1 by adding 99. 0% ultrapure acetic acid, pH 2 . 4. The solution was again centrifuged at 10, 000 gfor 30 min at 4C. == PHYSIQUE 2 . == Schematic diagram of downstream processing of recombinant protein GA733-FcK from plant leaf biomass. The second ammonium sulfate application (15, 30, 35, 40, 50, 60, 70, and 80%) was performed for TSP precipitation (the dotted.