Kisung Ko

Transgenic tomatoes expressing human beta-amyloid for use as a vaccine against Alzheimer’s disease

Human β-amyloid (Aβ) is believed to be one of the main components of Alzheimer’s disease, so reduction of Aβ is considered a key therapeutic target. Using Agrobacterium-mediated nuclear transformation, we generated transgenic tomatoes for Aβ with tandem repeats. Integration of the human Aβ gene into the tomato genome and its transcription were detected by PCR and Northern blot, respectively. Expression of the Aβ protein was confirmed by western blot and ELISA, and then the transgenic tomato line expressing the highest protein level was selected for vaccination. Mice immunized orally with total soluble extracts from the transgenic tomato plants elicited an immune response after receiving a booster. The results indicate that tomato plants may provide a useful system for the production of human Aβ antigen.

Role of genetic factors and environmental conditions in recombinant protein production for molecular farming.

Plants are generally considered to represent a promising heterologous expression system for the production of valuable recombinant proteins. Minimal upstream plant production cost is a salient feature driving the development of plant expression systems used for the synthesis of recombinant proteins. For such a plant expression system to be fully effective, it is first essential to improve plant productivity by plant biomass after inserting genes of interest into a suitable plant. Plant productivity is related closely to its growth and development, both of which are affected directly by environmental factors. These environmental factors that affect the cultivation conditions mainly include temperature, light, salinity, drought, nutrition, insects and pests. In addition, genetic factors that affect gene expression at the transcriptional, translational, and post-translational levels are considered to be important factors related to gene expression in plants. Thus, these factors influence both the quality and quantity of recombinant protein produced in transgenic plants. Among the genetic factors, the post-translational process is of particular interest as it influences subcellular localization, protein glycosylation, assembly and folding of therapeutic proteins, consequently affecting both protein quantity and biological quality. In this review, we discuss the effects of cultivation condition and genetic factors on recombinant protein production in transgenic plants.

Gangliosides are involved in neural differentiation of human dental pulp-derived stem cells

Human dental pulp-derived stem cells (hDPSCs) have been considered alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. This study investigated the possible role of gangliosides in the neural differentiation of hDPSCs. When hDPSCs were cultured under neural differentiation conditions, expression of neural cell marker genes such as Nestin, MAP-2, and NeuN was detected. Immunostaining and high-performance thin-layer chromatography analysis showed that an increase in ganglioside biosynthesis was associated with neural differentiation of hDPSCs. Specifically, a significant increase in GD3 and GD1a expression was observed during neural differentiation. To confirm the role of gangliosides in neural differentiation, ganglioside biosynthesis was inhibited in hDPSCs by knockdown of UDP-glucose ceramide glucosyltransferase (Ugcg), which prevented differentiation into neural cells. These results suggest that gangliosides may play a role in the neural differentiation process of hDPSCs.

High-throughput quantitative analysis of plant N-glycan using a DNA sequencer.

High-throughput quantitative analytical method for plant N-glycan has been developed. All steps, including peptide N-glycosidase (PNGase) A treatment, glycan preparation, and exoglycosidase digestion, were optimized for high-throughput applications using 96-well format procedures and automatic analysis on a DNA sequencer. The glycans of horseradish peroxidase with plant-specific core alpha(1,3)-fucose can be distinguished by the comparison of the glycan profiles obtained via PNGase A and F treatments. The peaks of the glycans with (91%) and without (1.2%) alpha(1,3)-fucose could be readily quantified and shown to harbor bisecting beta(1,2)-xylose via simultaneous treatment with alpha(1,3)-mannosidase and beta(1,2)-xylosidase. This optimized method was successfully applied to analyze N-glycans of plant-expressed recombinant antibody, which was engineered to contain a minor amount of glycan harboring beta(1,2)-xylose. These results indicate that our DNA sequencer-based method provides quantitative information for plant-specific N-glycan analysis in a high-throughput manner, which has not previously been achieved by glycan profiling based on mass spectrometry.

Effects of gangliosides on the differentiation of human mesenchymal stem cells into osteoblasts by modulating epidermal growth factor receptors

Gangliosides are sialic acid-conjugated glycosphingolipids that are believed to regulate cell differentiation as well as the signals of several signal molecules, including epidermal growth factor receptors (EGFR). These compounds are localized in a glycosphingolipid-enriched microdomain on the cell surface and regulated by the glycosphingolipid composition. However, the role that gangliosides play in osteoblastogenesis is not yet clearly understood, therefore, in this study, the relationship between gangliosides and EGFR activation was investigated during osteoblast differentiation in human mesenchymal stem cells (hMSCs). The results of high-performance thin-layer chromatography (HPTLC) showed that ganglioside GM3 expression was decreased, whereas ganglioside GD1a expression was increased during the differentiation of hMSCs into osteoblasts. In addition, an increase in the activation of alkaline phosphatase (ALP) was observed in response to treatment with EGF (5 ng/ml) and GD1a (1 microM) (p<0.05). The activation of ALP was significantly elevated in response to treatment of ganglioside GD1a with EGF when compared to control cells (p<0.01). However, treatment with GM3 (1muM) resulted in decreased ALP activation (p<0.01), and treatment of hMSCs with a chemical inhibitor of EGFR, AG1478, removed the differential effect of the two gangliosides. Moreover, incubation of the differentiating cells with GD1a enhanced the phosphorylation of EGFR, whereas treatment with GM3 reduced the EGFR phosphorylation. However, AG1478 treatment inhibited the effect of ganglioside GD1a elicitation on EGFR phosphorylation. Taken together, these results indicate that GD1a promotes osteoblast differentiation through the enhancement of EGFR phosphorylation, but that GM3 inhibits osteoblast differentiation through reduced EGFR phosphorylation, suggesting that GM3 and GD1a are essential molecules for regulating osteoblast differentiation in hMSCs.

Characterization of N-glycan structures and biofunction of anti-colorectal cancer monoclonal antibody CO17-1A produced in baculovirus-insect cell expression system

Advantages of the baculovirus insect cell expression system for production of recombinant proteins include high capacity, flexibility, and glycosylation capability. In this study, this expression system was exploited to produce anti-cancer monoclonal antibody (mAb) CO17-1A, which recognizes the antigen GA733. The heavy chain (HC) and light chain (LC) genes of mAb CO17-1A were cloned under the control of P(10) and Polyhedrin promoters in the pFastBac dual vector, respectively. Gene expression cassettes carrying the HC and LC genes were transposed into a bacmid in Escherichia coli (DH10Bac). The transposed bacmid was transfected to Sf9 insect cells to generate baculovirus expressing mAb CO17-1A. Confocal immunofluorescence and Western blot analyses confirmed expression of mAb CO17-1A in baculovirus-infected insect cells. The optimum conditions for mAb expression were evaluated at 24, 48, and 72 h after the virus infection at an optimum virus multiplicity of infection of 1. Expression of mAb CO17-1A in insect cells significantly increased at 72 h after infection. HPLC analysis of glycosylation status revealed that the insect-derived mAb (mAb(I)) CO17-1A had insect specific glycan structures. ELISA showed that the purified mAb(I) from cell culture supernatant specifically bound to SW948 human colorectal cancer cells. Fluorescence-activated cell sorting analysis showed that, although mAb(I) had insect specific glycan structures that differed from their mammalian counterparts, mAb(I) similarly interacted with CD64 (FcgammaRI) and Fc of IgG, compared to the interactions of mammalian-derived mAb. These results suggest that the baculovirus insect cell expression system is able to express, assemble, and secrete biofunctional full size mAb.

Oral administration of insulin-like growth factor-I from colostral whey reduces blood glucose in streptozotocin-induced diabetic mice

The aim of the present study was to investigate the effects of oral administration of the insulin-like growth factor-I-rich fraction (IGF-I-RF) from bovine colostral whey on the regulation of blood glucose levels in streptozotocin (STZ)-induced diabetic mice. We obtained a peptide fraction containing IGF-I (10 ng/mg protein) from Holstein colostrum within 24 h after parturition by using ultrafiltration. The blood glucose levels of STZ-induced diabetic mice fed with IGF-I-RF (50 μg/kg per d) were significantly reduced by 11 and 33 % at weeks 2 and 4, respectively (P < 0·05). The body weights of STZ-induced diabetic mice increased following the oral administration of the IGF-I-RF. The kidney weights of STZ-induced diabetic mice decreased significantly (P < 0·05) following the administration of the IGF-I-RF, and the liver weights of STZ-induced diabetic mice decreased significantly (P < 0·05) following the administration of 50 μg/kg per d of the IGF-I-RF. The present results indicate that the IGF-I-RF obtained from Holstein colostrum could be a useful component for an alternative therapeutic modality for the treatment of diabetes in insulin-resistant patients.

Optimization of in vitro Seed Germination of Taraxacum platycarpum

Dandelion (Taraxacum platycarpum) has been widely utilized for medicinal purposes. However, the dandelion seeds are relatively difficult to germinate under cultivation conditions, which hampers seedling propagation of dandelion plants and reduces the opportunity of usage of such a useful medicinal plant. Thus, in this study, in vitro conditions for the dandelion seed germination were optimized to enhance the germination rate. In seed washing steps, the sequential treatments with 20% of ethanol, 20% of NaOCl, and distilled water avoided microbial contamination with the highest in vitro germination rate (67.5%) from seeds sown in germination media. The media supplemented with 1.4 g/L of MS salts and 1% of sucrose significantly enhanced the germination rate compared to the media with 4.4 g/L of MS and 3% of sucrose. Sowing the seeds vertically in the optimized media supplement conditions, 1.4 g/L of MS salts and 1% of sucrose, gave the maximum in vitro germination rate (61%), which was almost three times higher than sowing seeds on a soil pot (23%). Our results indicate that the seed washing and sowing methods including germination medium supplements can be optimized to enhance in vitro seed germination of dandelion.

Expression of anti-breast cancer monoclonal antibody in transgenic plant

BACKGROUND: Plant expression system for mass production of recombinant proteins has several advantages over other existing expression systems with economical and safety issues. Breast cancer is a cancer originating from breast tissue and comprises almost 25% of all cancers in women world widely. Lewis-Y antigen is difucosylated oligosaccharide and is carried by glycoconjugates at cancer cell surface. In this study, the anti-breast cancer mAb BR55, which recognizes the epitope Lewis-Y, was expressed in the plant expression system. METHODS AND RESULTS: We have developed plant system for production of mAb BR55 with or without KDEL (the ER retention signal). This ER retention signal was attached to C-terminus of protein to help retain the recombinant glycoprotein carrying oligomannose glycans and enhance glycoprotein accumulation. PCR analysis was performed and confirmed the presence of recombinant genes. Western blot validated that the recombinant proteins mAb BR55 with or without KDEL were expressed in transgenic plants, moreover, the expression level of the mAb BR55 with KDEL was higher compared to the mAb BR55 without KDEL. CONCLUSION: These results indicate that KDEL fusion is a good way to produce proteins and plant can be an ideal expression system to obtain proteins and enhance accumulation of proteins.

Molecular cloning and expression patterns of FK506-binding protein 12, an immunophilin from the cabbage butterfly, Pieris rapae

FK506‐binding protein (FK506BP) class belonging to immunophilin protein family has been known to play key roles in modulating T‐cell activation, regulation of cell cycle and protein folding. However, little is known about the involvement of FK506BP during viral pathogenesis in insect host. In this study, an attempt has been made to focus on the involvement of FK506BP in antiviral innate immunity, by cloning the full‐length cDNA of FK506BP12 (PrFK506BP12) from the cabbage butterfly, Pieris rapae. It comprised of 532 bp (excluding poly‐A tail) with a longest open reading frame (ORF) of 327 bp encoding 108 amino acids. In silico analysis of PrFK506BP12 ORF revealed a highly conserved FK506‐binding domain (FKBD). As expected, it showed high homology to other FK506BPs identified from Bombyx mori (92%), Manduca sexta (91%), Suberites domuncula (82%), Tribolium castaneum (81%) and Aedes aegypti (74%). Expression of PrFK506BP12 was observed during developmental stages of P. rapae, but was pronounced in late pupal and adult stage. In addition, spatial expression pattern analysis indicated its high expression in the head and fat body. Furthermore, PrFK506BP12 mRNA was induced 12 h after LTA, Poly I:C treatment and 3h after Pieris rapae granulovirus (PrGV) treatment in carcass. It suggests that PrFK506BP12 appears to be involved in immune responses and also play an important role in the fat body, although it remains to be clarified about their precise role in response to granulovirus.