Production of Mature Recombinant Human Activin A in Transgenic Rice Cell Suspension Culture.

Activin A belongs to the transforming growth factor (TGF) family member, which exhibits a wide range of biological activities, including the regulation of cellular proliferation and differentiation and the promotion of neuronal survival. The isolation of AA from natural sources can only produce limited quantities of this bioactive protein. In this study, the whole gene of the precursor form of recombinant human activin A (rhAA) contains a signal peptide, and a pro-region and a mature region were cloned into an expression vector under the control of the rice α-amylase 3D (RAmy3D) promoter.

Systemic and Oral Immunogenicity of Porcine Epidemic Diarrhea Virus Antigen Fused to Poly-Fc of Immunoglobulin G and Expressed in ΔXT/FT Plants.

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family has become increasingly probelmatic in the pig farming industry. Currently, there are no effective, globally applicable vaccines against PEDV. Here, we tested a recombinant PEDV vaccine candidate based on the expression of the core neutralising epitope (COE) of PEDV conjugated to polymeric immunoglobulin G scaffold (PIGS) in glycoengineered plants.

Inactivation of the β (1, 2)-xylosyltransferase and the α (1, 3)-fucosyltransferase gene in rice (Oryza sativa) by multiplex CRISPR/Cas9 strategy.

CRISPR/Cas9-mediated OsXylT and OsFucT mutation caused the elimination of plant-specific β1,2-xylose and α1,3-fucose residues on glycoproteins in rice, which is the first report of OsXylT/OsFucT double KO mutation in rice. N-glycosylation pathway is the one of post-translational mechanism and is known as highly conserved in eukaryotes. However, the process for complex-N-glycan modification is different between mammals and plants.

Production of recombinant human acid β-glucosidase with high mannose-type N-glycans in rice gnt1 mutant for potential treatment of Gaucher disease.

Gaucher disease is an inherited metabolic disease caused by genetic acid β -glucosidase (GBA) deficiency and is currently treated by enzyme replacement therapy. For uptake into macrophages, GBA needs to carry terminal mannose residues on their N-glycans. Knockout mutant rice of N-acetylglucosaminyltransferase-I (gnt1) have a disrupted N-glycan processing pathway and produce only glycoproteins with high mannose residues.

Plant-expressed Fc-fusion protein tetravalent dengue vaccine with inherent adjuvant properties.

Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi Pasteur Dengvaxia vaccine does not protect children under the age of nine, and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant expression to produce a humanized and highly immunogenic poly-immunoglobulin G scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII).

Spontaneous pepsin C-catalyzed activation of human pepsinogen C in transgenic rice cell suspension culture: Production and characterization of human pepsin C.

A human pepsinogen C (hPGC) gene was synthesized with rice-optimized codon usage and cloned into a rice expression vector containing the promoter, signal peptide, and terminator derived from the rice α-amylase 3D (Ramy3D) gene. In addition, a 6-His tag was added to the 3' end of the synthetic hPGC gene for easy purification. The plant expression vector was introduced into rice calli (Oryza sativa L.

Molecular engineering and plant expression of an immunoglobulin heavy chain scaffold for delivery of a dengue vaccine candidate.

In order to enhance vaccine uptake by the immune cells in vivo, molecular engineering approach was employed to construct a polymeric immunoglobulin G scaffold (PIGS) that incorporates multiple copies of an antigen and targets the Fc gamma receptors on antigen-presenting cells. These self-adjuvanting immunogens were tested in the context of dengue infection, for which there is currently no globally licensed vaccine yet. Thus, the consensus domain III sequence (cEDIII) of dengue glycoprotein E was incorporated into PIGS and expressed in both tobacco plants and Chinese Ovary Hamster cells.

Production of recombinant human acid α-glucosidase with high-mannose glycans in gnt1 rice for the treatment of Pompe disease.

Lysosomal storage diseases are a group of inherited metabolic disorders. Patients are treated with enzyme replacement therapy (ERT), in which the replacement enzymes are required to carry terminal mannose or mannose 6-phosphate residues to allow efficient uptake into target cells and tissues. N-acetylglucosaminyltransferase-I (GnTI) mediates N-glycosylation in the cis cisternae of the Golgi apparatus by adding N-acetylglucosamine to the exposed terminal mannose residue of core N-glycan structures for further processing.

Oral immunisation of mice with transgenic rice calli expressing cholera toxin B subunit fused to consensus dengue cEDIII antigen induces antibodies to all four dengue serotypes.

Dengue virus (DENV) infection is an emerging global health threat. DENV consists of four distinct serotypes, necessitating a tetravalent vaccine. In this study, expression of consensus envelope protein domain III (cEDIII) fused to cholera toxin B subunit (CTB) in transgenic rice calli was improved using the luminal binding protein BiP at the N-terminus and the SEKDEL signal sequences at the C-terminus, targeting the recombinant protein to endoplasmic reticulum (ER).

Expression and assembly of cholera toxin B subunit and domain III of dengue virus 2 envelope fusion protein in transgenic potatoes.

The rates of mosquito-transmitted dengue virus infection in humans have increased in tropical and sub-tropical areas. Domain III of dengue envelope protein (EDIII) is involved in cellular receptor binding and induces serotype-specific neutralizing antibodies. EDIII fused to the B subunit of Vibrio cholera (CTB-EDIII) was expressed in potatoes to develop a plant-based vaccine against dengue virus type 2.

Production and immunogenicity of Actinobacillus pleuropneumoniae ApxIIA protein in transgenic rice callus.

Actinobacillus pleuropneumoniae is a major etiological agent that is responsible for swine pleuropneumonia, a highly contagious respiratory infection that causes severe economic losses in the swine production industry. ApxIIA is one of the virulence factors in A. pleuropneumoniae and has been considered as a candidate for developing a vaccine against the bacterial infection.

Production and characterization of recombinant human acid α-glucosidase in transgenic rice cell suspension culture.

Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid α-glucosidase (GAA), a glycogen-degrading lysosomal enzyme. In this study, the human GAA cDNA gene was synthesized from human placenta cells and cloned into a plant expression vector under the control of the rice α-amylase 3D (RAmy3D) promoter. The plant expression vector was introduced into rice calli (Oryza sativa L.

Immunogenicity of an S1D epitope from porcine epidemic diarrhea virus and cholera toxin B subunit fusion protein transiently expressed in infiltrated leaves.

Porcine epidemic diarrhea virus (PEDV) belongs to the family and causes acute enteritis in pigs. A fragment of the large spike glycoprotein, termed the S1D epitope (aa 636-789), alone and fused with cholera toxin B subunit, were independently cloned into plant expression vectors, yielding plasmids pMYV717 and pMYV719, respectively. Plant expression vectors were transformed into and subsequently infiltrated into leaves.

Novel vaccination approach for dengue infection based on recombinant immune complex universal platform.

Dengue infection is on the rise in many endemic areas of the tropics. Vaccination remains the most realistic strategy for prevention of this potentially fatal viral disease but there is currently no effective vaccine that could protect against all four known serotypes of the dengue virus. This study describes the generation and testing of a novel vaccination approach against dengue based on recombinant immune complexes (RIC).

Improved production of phleichrome from the phytopathogenic fungus Cladosporium phlei using synthetic inducers and photodynamic ROS production by phleichrome.

Two different diketopiperazines, cyclo-(L-Pro-L-Leu) and cyclo-(L-Pro-L-Phe), which were isolated from the culture filtrate of Epichloe typhina and found to be inducers of phleichrome production, were chemically synthesized and evaluated for use in the improved production of phleichrome from wild-type and UV-mutagenized strains (M0035) of Cladosporium phlei. When supplemented with PDA and V8 juice agar media, both inducers showed significant increases in the production of phleichrome. Phleichrome production was increased in a dose-dependent manner up to a concentration of maximum yield for both inducers.

Induction of immune responses in mice and pigs by oral administration of classical swine fever virus E2 protein expressed in rice calli.

Classical swine fever (CSF), caused by the CSF virus (CSFV), is a highly contagious disease in pigs. In Korea, vaccination using a live-attenuated strain (LOM strain) has been used to control the disease. However, parenteral vaccination using a live-attenuated strain still faces a number of problems related to storage, cost, injection stress, and differentiation of CSFV infected and vaccinated pigs.

Dengue virus E glycoprotein production in transgenic rice callus.

Dengue is a disease caused by dengue virus and represents the most important arthropod-borne viral disease in humans. Dengue virus enters host cells via binding of envelope glycoprotein (E) to a receptor. In this study, plant expression vectors containing native and synthetic glycoprotein E genes (sE) modified based on plant-optimized codon usage and fused with an ER retention signal were constructed under control of the rice amylase 3D promoter expression system.

Production of functional human vascular endothelial growth factor(165) in transgenic rice cell suspension cultures.

Vascular endothelial growth factors (VEGFs) are secreted by tumor cells and other cells exposed to hypoxia, and play a critical role in the development and differentiation of the vascular system. In this study, we investigated the production of functional recombinant human VEGF165 (rhVEGF165) in transgenic rice cell suspension culture. Complementary DNA was synthesized from human leukemia HL60 cells and cloned into expression vectors under the control of the rice α-amylase 3D (RAmy3D) promoter.

High-level production of recombinant trypsin in transgenic rice cell culture through utilization of an alternative carbon source and recycling system.

Productivity of recombinant bovine trypsin using a rice amylase 3D promoter has been studied in transgenic rice suspension culture. Alternative carbon sources were added to rice cell suspension cultures in order to improve the production of recombinant bovine trypsin. It was demonstrated that addition of alternative carbon sources such as succinic acid, fumaric acid and malic acid in the culture medium could increase the productivity of recombinant bovine trypsin 3.

Expression and characterization of an M cell-specific ligand-fused dengue virus tetravalent epitope using Saccharomyces cerevisiae.

A fusion construct (Tet-EDIII-Co1) consisting of an M cell-specific peptide ligand (Co1) at the C-terminus of a recombinant tetravalent gene encoding the amino acid sequences of dengue envelope domain III (Tet-EDIII) from four serotypes was expressed and tested for binding activity to the mucosal immune inductive site M cells for the development of an oral vaccine. The yeast episomal expression vector, pYEGPD-TER, which was designed to direct gene expression using the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, a functional signal peptide of the amylase 1A protein from rice, and the GAL7 terminator, was used to clone the Tet-EDIII-Co1 gene and resultant plasmids were then used to transform Saccharomyces cerevisiae. PCR and back-transformation into Escherichia coli confirmed the presence of the Tet-EDIII-Co1 gene-containing plasmid in transformants.

The identification of elephant ivory evidences of illegal trade with mitochondrial cytochrome b gene and hypervariable D-loop region.

DNA analysis of elephant ivory of illegal trade was handled in this work. The speciation and geographical origin of nine specimens of elephant ivory were requested by the police. Without national authorization, the suspect had purchased processed ivory seals from January to May, 2011 by Internet transactions from a site in a neighboring country.

Expression and purification of an immunogenic dengue virus epitope using a synthetic consensus sequence of envelope domain III and Saccharomyces cerevisiae.

A synthetic consensus gene was designed based on residues of the amino acid sequences of dengue envelope domain III (scEDIII) from all four serotypes, and codon optimization for expression was conducted using baker's yeast, Saccharomyces cerevisiae. The synthetic gene was cloned into a yeast episomal expression vector, pYEGPD-TER, which was designed to direct cloned gene expression using the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, a functional signal peptide of the amylase 1A protein from rice, and the GAL7 terminator. PCR and back-transformation into Escherichia coli confirmed the presence of the scEDIII gene-containing plasmid in the transformants.

M cell-targeting ligand and consensus dengue virus envelope protein domain III fusion protein production in transgenic rice calli.

The spread of dengue (DEN) virus is becoming a major concern due to the possibility of primary infection with one of the four dengue serotypes (DEN 1-4) and secondary infection with other heterotypes, which can further aggravate clinical manifestations. A gene encoding consensus envelope protein domain III (cEDIII) of dengue virus with neutralizing activity against four dengue virus serotypes was fused to M cell-targeting peptide ligand (Co1) to increase its mucosal immunogenicity and was introduced into rice calli under the control of the inducible rice amylase 3D promoter expression system. The integration and expression of scEDIII-Co1 fusion gene in transgenic rice calli were confirmed by genomic DNA PCR amplification, Northern and Western blot analyses, respectively.

Immunogenicity of a neutralizing epitope from porcine epidemic diarrhea virus: M cell targeting ligand fusion protein expressed in transgenic rice calli.

To increase immune responses of plant-based vaccines in intestine mucosal immune systems, a synthetic neutralizing epitope (sCOE) gene of porcine epidemic diarrhea virus (PEDV) was fused with M cell-targeting ligand (Co1) and introduced into a plant expression vector under the control of rice amylase 3D promoter. The sCOE-Co1 fusion gene was introduced into rice calli via the particle bombardment-mediated transformation method. The stable integration and transcriptional expression of the sCOE-Co1 fusion gene was confirmed by genomic DNA PCR amplification and Northern blot analysis, respectively.

Functional pentameric formation via coexpression of the Escherichia coli heat-labile enterotoxin B subunit and its fusion protein subunit with a neutralizing epitope of ApxIIA exotoxin improves the mucosal immunogenicity and protection against challenge by Actinobacillus pleuropneumoniae.

A coexpression strategy in Saccharomyces cerevisiae using episomal and integrative vectors for the Escherichia coli heat-labile enterotoxin B subunit (LTB) and a fusion protein of an ApxIIA toxin epitope produced by Actinobacillus pleuropneumoniae coupled to LTB, respectively, was adapted for the hetero-oligomerization of LTB and the LTB fusion construct. Enzyme-linked immunosorbent assay (ELISA) with GM1 ganglioside indicated that the LTB fusion construct, along with LTB, was oligomerized to make the functional heteropentameric form, which can bind to receptors on the mucosal epithelium. The antigen-specific antibody titer of mice orally administered antigen was increased when using recombinant yeast coexpressing the pentameric form instead of recombinant yeast expressing either the LTB fusion form or antigen alone.