Tae-Geum Kim

Assembly of cholera toxin B subunit full-length rotavirus NSP4 fusion protein oligomers in transgenic potato

A CTB-NSP4175 fusion gene encoding the entire 175-aa murine rotavirus NSP4 enterotoxin protein was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB-NSP4175 enterotoxin fusion gene was detected in the genomic DNA of transformed leaves by PCR DNA amplification. Synthesis and assembly of the full-length CTB-NSP4175 fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding of CTB-NSP4175 fusion protein pentamers to intestinal epithelial cell membrane receptors was quantified by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). The ELISA results showed that CTB-NSP4175 fusion protein was 0.006–0.026% of the total soluble tuber protein. The synthesis of CTB-NSP4175 monomers and their assembly into biologically active oligomers in transformed potato tubers demonstrates the feasibility of using edible plants for the synthesis of enterocyte-targeted full-length rotavirus enterotoxin antigens that retain all of their pathogenic epitopes for initiation of a maximum mucosal immune response.

Co-expression of proteinase inhibitor enhances recombinant human granulocyte–macrophage colony stimulating factor production in transgenic rice cell suspension culture

The synthetic gene (sPI-II) harboring the chymotrypsin (C1) and trypsin (T1) inhibitor domains of the Nicotiana alata serine proteinase inhibitor II gene has been previously expressed, and extracellular protease activity was shown to be reduced in the suspension culture medium. In this study, the sPI-II gene was introduced into transgenic rice cells expressing rhGM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor), in an effort to reduce protease activity and increase rhGM-CSF accumulation in the suspension culture medium. The integration and expression of the introduced sPI-II gene in the transgenic rice cells were verified via genomic DNA PCR amplification and Northern blot analysis, respectively. Relative protease activity was found to have been reduced and rhGM-CSF production was increased 2-fold in the co-transformed cell suspension culture with rhGM-CSF and the sPI-II gene, as compared with that observed in the transformed cell suspension culture expressing rhGM-CSF only. These results indicate that a transformed plant cell suspension culture system expressing the proteinase inhibitor can be a useful tool for increasing recombinant protein production.

Improvement of recombinant hGM-CSF production by suppression of cysteine proteinase gene expression using RNA interference in a transgenic rice culture

Recombinant proteins have been previously synthesized in a transgenic rice cell suspension culture system with the rice amylase 3D promoter, which can be induced via sugar starvation. However, the secreted recombinant proteins have been shown to be rapidly decreased as the result of proteolytic degradation occurring during prolonged incubation. The secreted proteases were identified via two-dimensional electrophoresis (2-DE) and ESI/Q-TOF mass spectrometry analyses. The internal amino acid sequences of 8 of 37 spots corresponded to cysteine proteinase (CysP), which is encoded for by Rep1 and EP3A. This result shows that CysP is a major secreted protease in rice cell suspension cultures following induction via sugar starvation. Intron-containing self-complementary hairpin RNA (ihpRNA)-mediated post-transcriptional gene silencing (PTGS) was applied to suppress the expression of CysP in rice cell suspension cultures. The reduction of rice CysP mRNA and the detection of siRNA specific to CysP, an initiator of RNAi, were verified via Northern blot analysis and RNase protection assays, respectively, thereby indicating that PTGS operated successfully in this system. The analysis of total secreted protease and CysP activities evidenced lower activity than was observed with the wild-type. Furthermore, suspension cultures of rice cells transformed with both hGM-CSF and the gene expressing the ihpRNA of CysP evidenced a reduction in total protease and CysP activities, and an up to 1.9-fold improvement in hGM-CSF production as compared to that observed in a rice cell line expressing hGM-CSF only. These results demonstrate the feasibility of the suppression of CysP via RNA interference to reduce protease activity and to increase target protein accumulation in rice cell suspension cultures.

Expression of a Cholera Toxin B Subunit-Neutralizing Epitope of the Porcine Epidemic Diarrhea Virus Fusion Gene in Transgenic Lettuce (Lactuca sativa L.)

Transgenic plants have been used as a safe and economic expression system for the production of edible vaccines. A synthetic cholera toxin B subunit gene (CTB) was fused with a synthetic neutralizing epitope gene of the porcine epidemic diarrhea virus (sCTB–sCOE), and the sCTB–sCOE fusion gene was introduced into a plant expression vector under the control of the ubiquitin promoter. This plant expression vector was transformed into lettuce (Lactuca sativa L.) using the Agrobacterium-mediated transformation method. Stable integration and transcriptional expression of the sCTB–sCOE fusion gene was confirmed using genomic DNA PCR analysis and northern blot analysis, respectively. The results of western blot analysis with anti-cholera toxin and anti-COE antibody showed the synthesis and assembly of CTB–COE fusion protein into oligomeric structures with pentameric sizing. The biological activity of CTB–COE fusion protein to its receptor, GM1-ganglioside, in transgenic plants was confirmed via GM1-ELISA with anti-cholera toxin and anti-COE antibody. Based on GM1-ELISA, the expression level of CTB–COE fusion proteins reached 0.0065% of the total soluble protein in transgenic lettuce leaf tissues. Transgenic lettuce successfully expressing CTB–COE fusion protein will be tested to induce efficient immune responses against porcine epidemic diarrhea virus infection by administration with raw material.

Synthesis of an HIV-1 Tat transduction domain-rotavirus enterotoxin fusion protein in transgenic potato.

A DNA fragment encoding a 12-amino acid (aa) HIV-1 Tat transduction peptide fused to a 90-aa murine rotavirus NSP4 enterotoxin protein (Tat-NSP490) was transferred to Solanum tuberosum by Agrobacterium tumefaciens-mediated transformation. The fusion gene was detected in the genomic DNA of transformed plant leaf tissues by PCR DNA amplification. The Tat-NSP490 fusion protein was identified in transformed tuber extracts by immunoblot analysis using anti-NSP490 and anti-Tat as the primary antibodies. Enzyme-linked immunosorbent assay results showed that the Tat-NSP490 fusion protein made up to 0.0015% of the total soluble tuber protein. The synthesis of Tat-NSP490 fusion protein in transformed potato tuber tissues demonstrates the feasibility of plant cell delivery of the HIV-1 Tat transduction domain as a carrier for non-specific targeting of fused antigens to the mucosal immune system.

Expression of a cholera toxin B subunit in transgenic lettuce (Lactuca sativa L.) using Agrobacterium-mediated transformation system

To increase expression level of cholera toxin B subunit (CTB) in lettuce plants, synthetic CTB (sCTB) gene based on the optimized codon usage was fused with an endoplasmic reticulum retention signal, KDEL. The sCTB gene was introduced into a plant expression vector and transformed to lettuce plants using Agrobacterium-mediated transformation system. As a selection marker, a bialaphos resistance (bar) gene that encodes phosphinothricin acetyltransferase (PAT), conferring tolerance to the herbicide phosphinothricin (PPT), was used. PCR amplification of genomic DNA confirmed the presence of the sCTB gene in the transgenic lettuce plants. Expressions of mRNA and protein of sCTB were observed by Northern and Western blot analyses, respectively. The sCTB synthesized in the transgenic lettuce showed strong affinity for GM1-ganglioside suggesting that the sCTB conserved the antigenic sites for binding and proper folding of pentameric CTB structure. The expression level of CTB was relatively high, reaching total soluble protein (TSP) levels of 0.24% in transgenic lettuce.

Cholera toxin B subunit-domain III of dengue virus envelope glycoprotein E fusion protein production in transgenic plants

Envelope glycoprotein E of the dengue virus, which plays a crucial role in its entry into host cells, has an immunogenic domain III (EIII, amino acids 297-394), which is capable of inducing neutralizing antibodies. However, mice immunized with EIII protein without adjuvant elicited low immune responses. To improve low immune responses, a DNA fragment, consisting of cholera toxin B subunit and EIII gene (CTB-EIII), was constructed and introduced into tobacco plant cells (Nicotiana tabacum L. cv. MD609) by Agrobacterium tumefaciens-mediated transformation methods. The integration and transcription of CTB-EIII fusion gene were confirmed in transgenic plants by genomic DNA PCR amplification and Northern blot analysis, respectively. The results of immunoblot analysis with anti-CTB and anti-dengue virus antibodies showed the expression of the CTB-EIII fusion protein in transgenic plant extracts. Based on the G(M1)-ELISA results, the CTB-EIII protein expressed in plants showed the biological activity for intestinal epithelial cell membrane glycolipid receptor, G(M1)-ganglioside, and its expression level was up to about 0.019% of total soluble protein in transgenic plant leaf tissues. The feasibility of using a plant-produced CTB-EIII fusion protein to generate immunogenicity against domain III will be tested in future animal experiments.

Synthesis and assembly of SIVmac Gag p27 capsid protein cholera toxin B subunit fusion protein in transgenic potato

A deoxyribonucleic acid (DNA) fragment encoding the cholera toxin B subunit (CTB) was linked 5′ to the simian immunodeficiency virus (SIVmac) Gag p27 capsid gene (CTB-Gag). The fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The CTB-Gag gene fusion was detected in transformed potato leaf genomic DNA by polymerase chain reaction-mediated DNA amplification. The results of immunoblot analysis with anti-CTB and anti-Gag antibodies verified the synthesis of biologically active CTB-Gag fusion protein in transformed leaf and tuber tissues. Synthesis and assembly of the CTB-Gag fusion protein into oligomeric structures of pentamer size was confirmed by GM1-ganglioside-enzyme-linked immunosorbent assay (GM1-ELISA) of transformed potato tuber tissue extracts. The binding of CTB-Gag fusion protein oligomers to intestinal epithelial cell membrane receptors quantified by GM1-ELISA showed that CTB-Gag fusion protein made up approx 0.016–0.022% of the total soluble tuber protein. The synthesis of CTB-Gag monomers and their assembly into biologically active CTB-Gag fusion protein oligomers in potato tuber tissues provides the opportunity for employment of the carrier and adjuvant properties of CTB for the development of edible plant-based subunit mucosal vaccines for enhanced mucosal immunity against SIV in macaques.

Synthesis and assembly of anthrax lethal factor-cholera toxin B-subunit fusion protein in transgenic potato.

A DNA encoding the 27-kDa domain I of anthrax lethal factor protein (LF), was linked to the carboxyl terminus of the cholera toxin B-subunit (CTB-LF). The CTB-LF fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated in vivo transformation methods and antibiotic-resistant plants were regenerated. The CTB-LF fusion gene was detected in transformed potato leaf genomic DNA by polymerase chain reaction (PCR)-mediated DNA amplification. Immunoblot analysis with anti-CTB and anti-LF primary antibodies verified the synthesis and assembly of biologically active CTB-LF fusion protein oligomers in transformed plant tuber tissues. Furthermore, the binding of CTB-LF fusion protein pentamers to intestinal epithelial cell membrane receptors measured by GM1-ganglioside enzymelinked immunosorbent assay (GM1-ELISA) indicated that the CTB-LF fusion protein made up approx 0.002% of the total soluble tuber protein. Synthesis of CTB-LF monomers and their assembly into biologically active CTB-LF fusion protein pentamers in potato tuber tissues demonstrates the feasibility of using edible plants for production and delivery of adjuvanted LF protein for CTB-mediated immunostimulation of mucosal immune responses against anthrax toxin.

Expression of a cholera toxin B subunit and consensus dengue virus envelope protein domain III fusion gene in transgenic rice callus

Dengue (DEN) is one of the most important emerging mosquito-borne viral human diseases. Therefore, an effective dengue vaccine with immune responses against all four dengue virus serotypes is highly needed. A fusion gene encoding a synthetic consensus envelope protein domain III (scEDIII) of dengue virus with neutralizing activity against the four dengue virus serotypes and with the B subunit of cholera toxin (CTB) to increase its mucosal immunogenicity was constructed and was introduced into rice callus under the control of the inducible rice amylase 3D promoter expression system. The integration and expression of the CTB-scEDIII fusion gene in transgenic rice callus were confirmed by genomic DNA PCR amplification, Northern, and Western blot analyses, respectively. The biological binding activity of the CTB-cEDIII fusion protein to its GM1-ganglioside receptor was confirmed via GM1-ELISA with anti-CT and anti-dengue virus antibodies. Delivery of the CTB-cEDIII fusion protein into mucosal immune inductive sites (including M cells) in BALB/c mice was confirmed by in vitro and in vivo antigen uptake assays. These results showed that the CTB-cEDIII fusion protein was produced in the transgenic rice callus, and that plant-produced ligand fusion antigen proteins have the potential to be targeted to the mucosal immune system for improvement of the overall immune responses.