Sequential Genome Editing and Induced Excision of the Transgene in BY2 Cells.

While plant cells in suspension are becoming a popular platform for expressing biotherapeutic proteins, the need to pre-engineer these cells to better comply with their role as host cell lines is emerging. Heterologous DNA and selectable markers are used for transformation and genome editing designated to produce improved host cell lines for overexpression of recombinant proteins. The removal of these heterologous DNA and selectable markers, no longer needed, can be beneficial since they limit additional gene stacking in subsequent transformations and may pose excessive metabolic burden on the cell machinery.

Immunogenicity of glycans on biotherapeutic drugs produced in plant expression systems-The taliglucerase alfa story.

Plants are a promising alternative for the production of biotherapeutics. Manufacturing in-planta adds plant specific glycans. To understand immunogenic potential of these glycans, we developed a validated method to detect plant specific glycan antibodies in human serum.

Establishment of a tobacco BY2 cell line devoid of plant-specific xylose and fucose as a platform for the production of biotherapeutic proteins.

Plant-produced glycoproteins contain N-linked glycans with plant-specific residues of β(1,2)-xylose and core α(1,3)-fucose, which do not exist in mammalian-derived proteins. Although our experience with two enzymes that are used for enzyme replacement therapy does not indicate that the plant sugar residues have deleterious effects, we made a conscious decision to eliminate these moieties from plant-expressed proteins. We knocked out the β(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes, using CRISPR/Cas9 genome editing, in Nicotiana tabacum L.

Insight into glucosidase II from the red marine microalga Porphyridium sp. (Rhodophyta).

N-glycosylation of proteins is one of the most important post-translational modifications that occur in various organisms, and is of utmost importance for protein function, stability, secretion, and loca-lization. Although the N-linked glycosylation pathway of proteins has been extensively characterized in mammals and plants, not much information is available regarding the N-glycosylation pathway in algae. We studied the α 1,3-glucosidase glucosidase II (GANAB) glycoenzyme in a red marine microalga Porphyridium sp.

Plant-based oral delivery of β-glucocerebrosidase as an enzyme replacement therapy for Gaucher's disease.

Gaucher's disease (GD), a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy (ERT) using recombinant GCD that is administered intravenously every 2 weeks. However, intravenous administration includes discomfort or pain and might cause local and systemic infections that may lead to low patient compliance. An orally administered drug has the potential to alleviate these problems.

Characterization of a chemically modified plant cell culture expressed human α-Galactosidase-A enzyme for treatment of Fabry disease.

Fabry disease is an X-linked recessive disorder caused by the loss of function of the lysosomal enzyme α-Galactosidase-A. Although two enzyme replacement therapies (ERTs) are commercially available, they may not effectively reverse some of the Fabry pathology. PRX-102 is a novel enzyme for the therapy of Fabry disease expressed in a BY2 Tobacco cell culture.

Genes involved in the endoplasmic reticulum N-glycosylation pathway of the red microalga Porphyridium sp.: a bioinformatic study.

N-glycosylation is one of the most important post-translational modifications that influence protein polymorphism, including protein structures and their functions. Although this important biological process has been extensively studied in mammals, only limited knowledge exists regarding glycosylation in algae. The current research is focused on the red microalga Porphyridium sp.

Glycosylation and functionality of recombinant β-glucocerebrosidase from various production systems.

The glycosylation of recombinant β-glucocerebrosidase, and in particular the exposure of mannose residues, has been shown to be a key factor in the success of ERT (enzyme replacement therapy) for the treatment of GD (Gaucher disease). Macrophages, the target cells in GD, internalize β-glucocerebrosidase through MRs (mannose receptors). Three enzymes are commercially available for the treatment of GD by ERT.

Unique N-glycan moieties of the 66-kDa cell wall glycoprotein from the red microalga Porphyridium sp.

We report here the structural determination of the N-linked glycans in the 66-kDa glycoprotein, part of the unique sulfated complex cell wall polysaccharide of the red microalga Porphyridium sp. Structures were elucidated by a combination of normal phase/reverse phase HPLC, positive ion MALDI-TOF MS, negative ion electrospray ionization, and MS/MS. The sugar moieties of the glycoprotein consisted of at least four fractions of N-linked glycans, each composed of the same four monosaccharides, GlcNAc, Man, 6-O-MeMan, and Xyl, with compositions Man(8-9)Xyl(1-2)Me(3)GlcNAc(2).

Serum apolipoproteins C-I and C-III are reduced in stomach cancer patients: results from MALDI-based peptidome and immuno-based clinical assays.

Finding new peptide biomarkers for stomach cancer in human sera that can be implemented into a clinically practicable prediction method for monitoring of stomach cancer. We studied the serum peptidome from two different biorepositories. We first employed a C8-reverse phase liquid chromatography approach for sample purification, followed by mass-spectrometry analysis.

A pivotal role for the Streptococcus iniae extracellular polysaccharide in triggering proinflammatory cytokines transcription and inducing death in rainbow trout.

Streptococcus iniae is a major pathogen of fish, causing considerable economic losses in Israel, the United States and the Far East. Containment of mortalities through vaccination was recently compromised due to the emergence of novel vaccine-escape strains that are distinguished from previous strains by their ability to produce large amounts of extracellular polysaccharide (EPS) that is released to the medium. In vitro and in vivo data now indicate that the EPS is a major virulence factor, capable of triggering the proinflammatory cytokine machinery and inducing mortality of fish.

NKp46 O-glycan sequences that are involved in the interaction with hemagglutinin type 1 of influenza virus.

Natural killer (NK) cells serve as a crucial first-line defense against tumors and virus-infected cells. We previously showed that lysis of influenza virus (IV)-infected cells is mediated by the interaction between the NK receptor, NKp46, and the IV hemagglutinin (HA) type 1 expressed by the infected cells. This interaction requires the presence of sialyl groups on the NKp46-T225 O-glycoforms.

Chemical characterization, antiproliferative and antiadhesive properties of polysaccharides extracted from Pleurotus pulmonarius mycelium and fruiting bodies.

Mushroom polysaccharides are potent substances that exhibit antitumor and immunomodulatory properties. Studies comparing the chemical composition and antitumor-related activities of polysaccharides released by fungal strains under different growth conditions are not available. Thus, the present study compared polysaccharides extracts produced by Pleurotus pulmonarius from mycelium grown in liquid culture (ME) or fruiting bodies (FBE).

Isolation and characterization of poly- and oligosaccharides from the red microalga Porphyridium sp.

The current study forms part of an ongoing research effort focusing on the elucidation of the chemical structure of the sulfated extracellular polysaccharide of the red microalga Porphyridium sp. (UTEX 637). We report here on the chemical structure of a fraction separated from an acidic crude extract of the polysaccharide, as investigated by methylation analysis, carboxyl reduction-methylation analysis, desulfation-methylation analysis, partial acid hydrolysis, Smith degradation, together with 1D and 2D (1)H and (13)C NMR spectroscopy.

Emergence of novel Streptococcus iniae exopolysaccharide-producing strains following vaccination with nonproducing strains.

Streptococcus iniae is a major pathogen of fish, producing fatal disease among fish species living in very diverse environments. Recently, reoccurrences of disease outbreaks were recorded in rainbow trout (Oncorhynchus mykiss, Walbaum) farms where the entire fish population was routinely vaccinated. New strains are distinguished from previous strains by their ability to produce large amounts of extracellular polysaccharide that is released into the medium.

Altered glycosylation of recombinant NKp30 hampers binding to heparan sulfate: a lesson for the use of recombinant immunoreceptors as an immunological tool.

NKp30 is a natural cytotoxicity receptor expressed by human NK cells and involved in NK lytic activity. We previously published that membranal heparan sulfate serves as a coligand for human NKp30. In the present study, we complement our results by showing direct binding of recombinant NKp30 to immobilized heparin.

Effect of organic and inorganic nitrogenous compounds on RDX degradation and cytochrome P-450 expression in Rhodococcus strain YH1.

We hypothesized that biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)--a widely used explosive contaminating soil and groundwater--by Rhodococcus strain YH1 is controlled by the presence of external nitrogen sources. This strain is capable of degrading RDX while using it as sole nitrogen source under aerobic conditions. Both inorganic and organic nitrogen sources were found to have a profound impact on RDX-biodegradation activity.

Controlled glycosylation of therapeutic antibodies in plants.

Recombinant therapeutic monoclonal antibodies (mAb) can be expressed, assembled, and glycosylated in plants. Transgenic plants, producing anti-rabies mAb and anti-colorectal cancer mAb, were obtained from Agrobacterium-mediated transformation. The heavy chain (HC) of anti-rabies mAb was fused to the Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum retention signal whereas the HC of anti-colorectal cancer mAb was not fused to the KDEL sequence.

Function and glycosylation of plant-derived antiviral monoclonal antibody.

Plant genetic engineering led to the production of plant-derived mAb (mAbP), which provides a safe and economically feasible alternative to the current methods of antibody production in animal systems. In this study, the heavy and light chains of human anti-rabies mAb were expressed and assembled in planta under the control of two strong constitutive promoters. An alfalfa mosaic virus untranslated leader sequence and Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum retention signal were linked at the N and C terminus of the heavy chain, respectively.