The tumor-associated antigen GA733 is a cell-surface glycoprotein highly expressed in colorectal carcinomas. In this study, 3 recombinant genes were constructed as follows: GA733 tagged to the ER retention sequence KDEL (GA733K), GA733 fused to the immunoglobulin Fc fragment (GA733-Fc), and GA733-Fc fused to the ER retention sequence (GA733-FcK). Agrobacterium-mediated transformation was used to generate transgenic plants expressing recombinant genes. The presence of transgenes was confirmed by genomic PCR. Western blot, confocal immunofluorescence, and sandwich ELISA showed the expression of recombinant proteins. The stability, flexibility, and bioactivity of recombinant proteins were analyzed and demonstrated through N-glycosylation analysis, animal trials, and sera ELISA. Our results suggest that the KDEL retained proteins in ER with oligomannose glycan structure and enhanced protein accumulation level. The sera of mice immunized with GA733-FcK purified from plants contained immunoglobulins which were at least as efficient as the mammalian-derived GA733-Fc at recognizing human colorectal cancer cell lines. Thus, a plant system can be used to express the KDEL fusion protein with oligomannose glycosylation, and this protein induces an immune response which is comparable to non-KDEL-tagged, mammalian-derived proteins.

Plant genetic engineering, which has led to the production of plant-derived monoclonal antibodies (mAbPs), provides a safe and economically effective alternative to conventional antibody expression methods. In this study, the expression levels and biological properties of the anti-rabies virus mAbP SO57 with or without an endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) in transgenic tobacco plants (Nicotiana tabacum) were analyzed. The expression levels of mAbP SO57 with KDEL (mAbPK) were significantly higher than those of mAbP SO57 without KDEL (mAbP) regardless of the transcription level. The Fc domains of both purified mAbP and mAbPK and hybridoma-derived mAb (mAbH) had similar levels of binding activity to the FcγRI receptor (CD64). The mAbPK had glycan profiles of both oligomannose (OM) type (91.7%) and Golgi type (8.3%), whereas the mAbP had mainly Golgi type glycans (96.8%) similar to those seen with mAbH. Confocal analysis showed that the mAbPK was co-localized to ER-tracker signal and cellular areas surrounding the nucleus indicating accumulation of the mAbP with KDEL in the ER. Both mAbP and mAbPK disappeared with similar trends to mAbH in BALB/c mice. In addition, mAbPK was as effective as mAbH at neutralizing the activity of the rabies virus CVS-11. These results suggest that the ER localization of the recombinant mAbP by KDEL reprograms OM glycosylation and enhances the production of the functional antivirus therapeutic antibody in the plant.

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The baculovirus-insect cell system is considered a feasible expression system for recombinant glycoprotein production due to its several advantages, including high capacity, flexibility, and glycosylation capability. However, accurate titering of the recombinant baculovirus is required to ensure high expression in insect cells using a commercial and expensive immunoassay titer kit in which the envelope glycoprotein of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-type baculovirus is detected by anti-envelope glycoprotein antibody and a secondary antibody conjugated to horseradish peroxidase (HRP). In this study, conditions for the expression of the CO17-1A immunotherapeutic monoclonal antibody (MAb) against colorectal cancer cells in a baculovirus system were optimized without using a commercial titering kit. Several variables were investigated to optimize antibody expression in a baculovirus-insect cell system, including baculovirus passage, volume of the infecting baculovirus inoculum (100, 200, 400, and 800 μL), and the harvest time of insect cells or cell supernatants after virus infection (24, 48, and 72 h). Two different pFastBac vectors carrying the CO17-1A MAb genes with or without the KDEL endoplasmic reticulum (ER) retention motif (Lys-Asp-Glu-Leu) fused to the HC (MAb CO17-1A K and MAb CO17-1A, respectively) were constructed and used to generate baculoviruses. Immunoblot analysis was conducted to confirm expression of MAb CO17-1A K and MAb CO17-1A in baculovirus-infected insect cells. Densitometry analysis of the protein bands was used to quantify the relative expression under different conditions. The highest expression was observed in lysed cells infected with 400 μL of passage 3 baculovirus (P(3) BV) carrying the gene encoding the CO17-1A MAb without KDEL at 72 h after virus infection. These results suggest that the infection conditions, the number of virus passages, baculovirus inoculum volume, and the harvest time can be modified to optimize MAb expression without using a BaculoELISA titer kit in a baculovirus-insect cell system.

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