The endoplasmic reticulum (ER) is the main site of protein synthesis, folding, and secretion to other organelles. The capacity of the ER to process proteins is limited, and excessive accumulation of unfolded and misfolded proteins can induce ER stress, which is associated with plant diseases. Here, a transgenic Arabidopsis system was established to express anti-cancer monoclonal antibodies (mAbs) that recognize the tumor-associated antigen GA733-2. Monoclonal antibody (mAb) CO17-1A recognize a tumor-associated epitope expressed on the colorectal cancer cell surface. The ER retention Lys-Asp-Glu-Leu (KDEL) motif sequence was added to the C-terminus of the heavy chain to retain anti-colorectal cancer mAbs in the ER, consequently boosting mAb production. Agrobacterium-mediated floral dip transformation was used to generate T1 transformants, and homozygous T4 seeds obtained from transgenic Arabidopsis plants expressing anti-colorectal cancer mAbs were used to confirm the physiological effects of KDEL tagging. Germination rates were not significantly different between both plants expressing mAb CO without KDEL mAb CO (CO plant) and mAb CO with KDEL mAb COK (COK plant). However, COK plants primary root lengths were shorter than those of CO plants and non-transgenic Arabidopsis plants in in vitro media. Most ER stress-related genes, with the exception of bZIP28 and IRE1a, were upregulated in COK plants compared to CO plants. Western blot and SDS-PAGE analyses showed that COK plants exhibited up to five times higher expression and mAb amounts than plants. Enhanced expression in mAb COK plants was confirmed by immunohistochemical analyses. mAb COK was distributed across most of the area of leaf tissues, whereas mAb CO was mainly distributed in extracellular areas. Surface plasmon resonance analyses revealed that mAb CO and mAb COK possessed equivalent or slightly better binding activities to antigen EpCAM compared to a commercially available parental antibody. N-glycosylation analysis showed that mAb CO had plant specific residues whereas mAb COK mainly showed an oligo-mannose N-glycan structure without the plant specific glycan residues. In this study, the reduction of plant growth and biomass induced by ER retention signal peptide might be only in in vitro conditions, and thus should be carefully considered for the initial screening for transgenic lines on culture media. Taken together, nevertheless the fusion of ER retention signal peptide is an effective approach for enhancing the yields of recombinant proteins in vivo.

The expression and glycosylation patterns of anti-colorectal cancer therapeutic monoclonal antibody (mAb) CO17-1A recognizing the tumor-associated antigen GA733-2, expressed in human colorectal carcinoma cells, were observed in the leaf and stem tissues of primary (0 cycle), secondary (1 cycle), and tertiary (2 cycle) growths of seedlings obtained from the stem cut of T2 plants. The bottom portion of the stem of T2 seedlings was cut to induce the 1 cycle shoot growth, which was again cut to induce the 2 cycle shoot growth. In the 1 and 2 cycle growths, the periods for floral organ formation (35 days) was shorter than that (100 days) for the 0 cycle growth. The genes of heavy and light chains of mAb CO17-1A existed at the top, middle, and basal portions of the leaves and stem obtained from the 0, 1, and 2 cycle plants. The protein levels in the leaves and stem tissues from the 1 and 2 cycles were similar to those in the tissues from the 0 cycle. The glycosylation level and pattern in the leaf and stem did not alter dramatically over the different cycles. Surface plasmon resonance (SPR) confirmed that mAbs CO17-1A obtained from leaf and stem tissues of the 0, 1, and 2 cycles had similar binding affinity for the GA733-2 antigen. These data suggest that the shoot growth by bottom stem cutting is applicable to speed up the growth of plant biomass expressing anti-colorectal cancer mAb without variation of expression, glycosylation, and functionality.

Various horticultural crops have been considered as potential heterologous expression systems for plant-made recombinant pharmaceutical proteins. However, there is little information concerning the total soluble protein (TSP) levels of major horticultural crops. Ten major horticultural crops-Chinese cabbage, broccoli, garlic, onion, cabbage, bunching onion, cucumber, zucchini, radish, and carrot, along with tobacco and Arabidopsis, were selected to investigate the TSP levels in their individual tissues. In tobacco, SDS-PAGE assay showed that leaf tissues had stronger protein bands than stem tissues, and freshly harvested samples had slightly stronger band density than the -70°C frozen samples, suggesting that fresh leaf should be used to measure the total soluble proteins without any protein loss or degradation. Bicinchoninic acid (BCA) protein assay revealed that among various horticultural crops, garlic (41.4 mg·g -1 FW), broccoli (21.9 mg·g -1 FW), and Chinese cabbage (11.9 mg·g -1 FW) had the highest TSP levels suggesting that these horticultural crops could be good candidates for plant molecular biofarming to produce highly valuable recombinant proteins. The inner clove tissue in garlic, the flower tissue in broccoli, and the green leaf tissue in Chinese cabbage showed the strongest protein band density as compared to other tissues. The TSP of Arabidopsis tissues was quantified by SDS-PAGE, BCA, and Nano-drop methods. In general, the middle leaf tissue showed the highest TSP levels. To evaluate TSP levels of various horticultural crops, these three different methods were compared. The correlation and regression analyses between SDS-PAGE and BCA, and SDS-PAGE and Nano-drop suggested that there were significant correlations between SDS-PAGE and BCA protein assays as compared to SDS-PAGE and Nano-drop assays, indicating that BCA assay is reliable to quantify TSP levels. In conclusion, the TSP levels varied depending on the horticultural crops and their tissue types, and BCA assay could be applied to quantify the TSP.Additional key words: BCA protein assay, nano-drop, protein quantification method, SDS-PAGE, total soluble protein (TSP)Hort.

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Circulating tumor cells (CTCs) are an indicator of metastatic progression and relapse. Since non-CTC cells such as red blood cells outnumber CTCs in the blood, the separation and enrichment of CTCs is key to improving their detection sensitivity. The ATP luminescence assay can measure intracellular ATP to detect cells quickly but has not yet been used for CTC detection in the blood because extracellular ATP in the blood, derived from non-CTCs, interferes with the measurement. Herein, we report on the improvement of the ATP luminescence assay for the detection of CTCs by separating and concentrating CTCs in the blood using a 3D printed immunomagnetic concentrator (3DPIC). Because of its high-aspect-ratio structure and resistance to high flow rates, 3DPIC allows cancer cells in 10 mL to be concentrated 100 times within minutes. This enables the ATP luminescence assay to detect as low as 10 cells in blood, thereby being about 10 times more sensitive than when commercial kits are used for CTC concentration. This is the first time that the ATP luminescence assay was used for the detection of cancer cells in blood. These results demonstrate the feasibility of 3DPIC as a concentrator to improve the detection limit of the ATP luminescence assay for the detection of CTCs.