Removal of plant endogenous proteins from tobacco leaf extract by freeze-thaw treatment for purification of recombinant proteins.

Plants are useful as a low-cost source for producing biopharmaceutical proteins. A significant hurdle in the production of recombinant proteins in plants, however, is the complicated process of removing plant-derived components. Removing endogenous plant proteins, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a major photosynthetic plant enzyme that catalyzes photosynthesis through carboxylation and oxygenation, is important for the purification of recombinant plant proteins. In particular, RuBisCO accounts for 50% of the soluble leaf protein; thus, the removal of RuBisCO is critical for the purification of recombinant proteins from plant materials. An effective conventional method, known as freeze-thaw treatment, was developed for the removal of RuBisCO from Nicotiana benthamiana, which expresses recombinant green fluorescent protein (GFP). Crude extracts or supernatants were frozen at - 30 °C. Upon thawing, most of the RuBisCO was precipitated by centrifugation without significant inactivation and/or yield reduction of GFP. Based on the proteomics analysis, using this method, RuBisCO large and small subunits were reduced to approximately 10% and 20% of those of the unfrozen supernatant solutions, respectively, without the need for specific reagents or equipment. The proteomic analysis also revealed that many ribosomal proteins were removed from the extracts. This method improves the purification process of recombinant proteins from plant materials. Prolonged freezing damaged recombinant ß-glucuronidase (GUS), suggesting that the applicability of this treatment should be carefully considered for each recombinant protein.

Herpes zoster peripheral nerve complications: Their pathophysiology in spinal ganglia and nerve roots.

Varicella zoster virus (VZV) causes chickenpox at the primary infection and then becomes latent in the spinal dorsal root ganglia; VZV can reactivate with aging, immunosuppression, stress, and other factors, occurring as herpes zoster (HZ) at 1-2 skin segments. HZ peripheral nerve complications caused by VZV reactivation include Hunt syndrome, segmental HZ paresis, post-herpetic neuralgia, and Guillain-Barré syndrome (GBS). We have encountered the rare HZ complications of upper-limb paresis, myeloradiculitis, and polyradiculoneuritis: an adult woman with upper-limb paresis consistent with the nerve root on segments above the thoracic HZ dermatome; another woman exhibiting ascending myeloradiculitis originating at the Th11-12 roots; an elderly woman with ascending VZV polyradiculoneuritis resembling GBS; an adult with VZV quadriplegia with disseminated HZ; and an elderly patient with VZV-associated polyradiculoneuritis. The three polyradiculoneuritis cases may be a new subtype of HZ peripheral neuropathy, but the pathophysiology for these HZ peripheral nerve complications unrelated to HZ dermatomes is unclear. We analyzed host factors, skin lesions, neurological and virological findings, and MRI results including 3D NerveVIEW in 15 Japanese patients treated at our facility for HZ peripheral neuropathy, including six differing from the HZ dermatome. Based on the clinical findings including MRI results of spinal ganglia and roots, we identified four possible routes for the patterns of VZV spread: (i) ascending spinal roots, (ii) ascending spinal cord, (iii) polyradiculopathy, and (iv) intrathecal spread. The incidence of HZ is increasing with the aging of many populations, and clinicians should be aware of these HZ neuropathies.

Seed transmission of raspberry bushy dwarf virus is blocked in Nicotiana benthamiana plants by preventing virus entry into the embryo from the infected embryo sac and endosperm.

Raspberry bushy dwarf virus (RBDV) is transmitted through seed in infected red raspberry plants after pollination with pollen grains from healthy red raspberry plants. Here, we show that RBDV is not transmitted through seeds in infected Nicotiana benthamiana (Nb) plants after pollination with virus-free Nb pollen grains. Chromogenic in situ hybridization revealed that the virus invades the shoot apical meristem and the ovule, including the embryo sac, of RBDV-infected Nb plants; however, in seeds that developed from infected embryo sacs after fertilization by virus-free sperm cells, RBDV was absent in the embryos and present in the endosperms. When we analyzed seed transmission of RBDV in Nb mutants with mutations in dicer-like enzyme 2 and 4 (NbDCL2&4) or RNA-dependent RNA polymerase 6 (NbRDR6), RBDV was not present in the offspring from seeds with embryos and endosperms that did not express NbDCL2&4 or NbRDR6. These results suggest that seed transmission of RBDV is prevented by evasion of infection by the embryo and that RNA silencing is not essential for preventing seed transmission of RBDV in Nb plants.

CRISPR/Cas9-mediated knockout of the DCL2 and DCL4 genes in Nicotiana benthamiana and its productivity of recombinant proteins.

KEY MESSAGE: DCL2 and DCL4 genes in Nicotiana benthamiana plants were successfully edited using the CRISPR/Cas9 system. Recently, plants have been utilized for recombinant protein production similar to other expression systems, i.e., bacteria, yeast, insect, and mammal cells. However, insufficient amounts of recombinant proteins are often produced in plant cells. The repression of RNA silencing within plant cells could improve production levels of recombinant protein because RNA silencing frequently decomposes mRNAs from transgenes. In this study, the genes dicer-like protein 2 and 4 (NbDCL2 and NbDCL4) were successfully edited to produce double-knockout transgenic Nicotiana benthamiana plants (dcl2dcl4 plants) using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. A transient green fluorescent protein (GFP) gene expression assay revealed that the dcl2dcl4 plants accumulated higher amounts of GFP and GFP mRNA than wild type (WT) and RNA-dependent RNA polymerase 6-knockout N. benthamiana plants (ΔRDR6 plants). Small RNA sequencing also showed that dcl2dcl4 plants accumulated lower amounts of small interfering RNAs (siRNAs) against the GFP gene than WT plants. The dcl2dcl4 plants might also produce higher amounts of human fibroblast growth factor 1 (FGF1) than WT and ΔRDR6 plants. These observations appear to reflect differences between DCLs and RDR6 in plant cell biological mechanisms. These results reveal that dcl2dcl4 plants would be suitable as platform plants for recombinant protein production.

Evaluation of methods for plant genomic DNA sequence analysis without DNA and PCR product purification.

Genome-editing technologies are widely used to characterize gene functions and improve the features of agricultural plants. Although sequence analysis of gene editing target DNA is the most reliable method of screening gene-edited plants, the current DNA sequence analysis methods are time consuming and labor intensive because they include genomic DNA and polymerase chain reaction (PCR) product purification. In this study, seven methods were performed for sequence analysis of plant genomic DNA with and/or without genomic DNA and PCR product purification. Consequently, good-quality sequencing chromatograms were obtained using all methods. Results showed that the partial genomic DNA sequence of Nicotiana benthamiana and Arabidopsis thaliana could be sufficiently analyzed without plant genomic DNA and PCR product purification. Furthermore, screening of gene-edited N. benthamiana was successful using the present methods. Therefore, the tested methods could reduce the time, simplify the workflow of plant gene analysis, and help in screening gene-edited plants.

Virus-Mediated Targeted DNA Methylation Illuminates the Dynamics of Methylation in an Endogenous Plant Gene.

DNA methylation maintains genome stability and regulates gene expression in plants. RNA-directed DNA methylation (RdDM) is critical for appropriate methylation. However, no efficient tools are available for the investigation of the functions of specific DNA methylation. In this study, the cucumber mosaic virus vector was used for targeted DNA methylation. Methylation was rapidly induced but gradually decreased from the 3' end of the target endogenous sequence in Nicotiana benthamiana, suggesting a mechanism to protect against the ectopic introduction of DNA methylation. Increasing 24-nt siRNAs blocked this reduction in methylation by down-regulating DCL2 and DCL4. RdDM relies on the sequence identity between RNA and genomic DNA; however, this identity does not appear to be the sole determinant for efficient DNA methylation. The current findings provide new insight into the regulation of DNA methylation and promote additional effort to develop efficient targeted DNA methylation in plants.

CRISPR/Cas9-mediated knockout of the RDR6 gene in Nicotiana benthamiana for efficient transient expression of recombinant proteins.

MAIN CONCLUSION: RDR6 gene knockout Nicotiana benthamiana plant was successfully produced using CRISPR/Cas9 technology. The production of recombinant proteins in plants has many advantages, such as safety and reduced costs. However, there are several problems with this technology, especially low levels of protein production. The dysfunction of the RNA silencing mechanism in plant cells would be effective to improve recombinant protein production because the RNA silencing mechanism efficiently degrades transgene-derived mRNAs. Therefore, to overcome this problem, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology was used to develop RNA silencing-related gene knockout transgenic Nicotiana benthamiana. We successfully produced RNA-dependent RNA polymerase 6 (RDR6), one of the most important components of the RNA silencing mechanism-knockout N. benthamiana (ΔRDR6 plants). The ΔRDR6 plants had abnormal flowers and were sterile, as with the Arabidopsis RDR6 mutants. However, a transient gene expression assay showed that the ΔRDR6 plants accumulated larger amounts of green fluorescent protein (GFP) and GFP mRNA than the wild-type (WT) plants. Small RNA sequencing analysis revealed that levels of small interfering RNA against the GFP gene were greatly reduced in the ΔRDR6 plants, as compared to that of the WT plants. These findings demonstrate that the ΔRDR6 plants can express larger amounts of recombinant proteins than WT plants and, therefore, would be useful for recombinant protein production and understanding the contributions of RDR6 to genetic and physiological events in plants.

Xylosylation of proteins by expression of human xylosyltransferase 2 in plants.

Through the years, the post-translational modification of plant-made recombinant proteins has been a considerable problem. Protein glycosylation is arguably the most important post-translational modification; thus, for the humanization of protein glycosylation in plants, the introduction, repression, and knockout of many glycosylation-related genes has been carried out. In addition, plants lack mammalian-type protein O-glycosylation pathways; thus, for the synthesis of mammalian O-glycans in plants, the construction of these pathways is necessary. In this study, we successfully xylosylated the recombinant human proteoglycan core protein, serglycin, by transient expression of human xylosyltransferase 2 in Nicotiana benthamiana plants. When human serglycin was co-expressed with human xylosyltransferase 2 in plants, multiple serine residues of eight xylosylation candidates were xylosylated. From the results of carbohydrate assays for total soluble proteins, some endogenous plant proteins also appeared to be xylosylated, likely through the actions of xylosyltransferase 2. The xylosylation of core proteins is the initial step of the glycosaminoglycan part of the synthesis of proteoglycans. In the future, these novel findings may lead to whole mammalian proteoglycan synthesis in plants.

Repression of the DCL2 and DCL4 genes in Nicotiana benthamiana plants for the transient expression of recombinant proteins.

The production of recombinant proteins in plants has many advantages, including safety and reduced costs. However, this technology still faces several issues, including low levels of production. The repression of RNA silencing seems to be particularly important for improving recombinant protein production because RNA silencing effectively degrades transgene-derived mRNAs in plant cells. Therefore, to overcome this, we used RNA interference technology to develop DCL2- and DCL4-repressed transgenic Nicotiana benthamiana plants (ΔD2, ΔD4, and ΔD2ΔD4 plants), which had much lower levels of NbDCL2 and/or NbDCL4 mRNAs than wild-type plants. A transient gene expression assay showed that the ΔD2ΔD4 plants accumulated larger amounts of green fluorescent protein (GFP) and human acidic fibroblast growth factor (aFGF) than ΔD2, ΔD4, and wild-type plants. Furthermore, the levels of GFP and aFGF mRNAs were also higher in ΔD2ΔD4 plants than in ΔD2, ΔD4, and wild-type plants. These findings demonstrate that ΔD2ΔD4 plants express larger amounts of recombinant proteins than wild-type plants, and so would be useful for recombinant protein production.

A simple agroinfiltration method for transient gene expression in plant leaf discs.

In the present study, we developed a simple transient gene expression system based on Agrobacterium-mediated transformation. Vacuum infiltration was applied to leaf discs from Nicotiana benthamiana plants with Agrobacterium suspension solution under conventional vacuum conditions in a needleless plastic syringe. Model proteins, green fluorescent protein, ß-glucuronidase, mouse granulocyte-macrophage colony-stimulating factor, and human fibroblast growth factor 1 were successfully expressed in leaf discs within 4 days after infiltration. In addition, the functional evaluation of viral RNA silencing suppressors, Artichoke mottled crinkle virus p19 protein, was also performed. Using this method, the contamination and diffusion of genetically modified bacterium to the environment and important transgenic plants were prevented. This method can be conducted without specialized apparatuses or large amounts of Agrobacterium suspension solutions; thus, the simultaneous evaluation of multiple vectors will be easily possible.

NMR-based structural validation of therapeutic antibody produced in Nicotiana benthamiana.

KEY MESSAGE: We successfully developed a method for metabolic isotope labeling of recombinant proteins produced in transgenic tobacco. This enabled assessment of structural integrity of plant-derived therapeutic antibodies by NMR analysis. A variety of expression vehicles have been developed for the production of promising biologics, including plants, fungi, bacteria, insects, and mammals. Glycoprotein biologics often experience altered folding and post-translational modifications that are typified by variant glycosylation patterns. These differences can dramatically affect their efficacy, as exemplified by therapeutic antibodies. However, it is generally difficult to validate the structural integrity of biologics produced using different expression vehicles. To address this issue, we have developed and applied a stable-isotope-assisted nuclear magnetic resonance (NMR) spectroscopy method for the conformational characterization of recombinant antibodies produced in plants. Nicotiana benthamiana used as a vehicle for the production of recombinant immunoglobulin G (IgG) was grown in a (15)N-enriched plant growth medium. The Fc fragment derived from the (15)N-labeled antibody thus prepared was subjected to heteronuclear two-dimensional (2D) NMR measurements. This approach enabled assessment of the structural integrity of the plant-derived therapeutic antibodies by comparing their NMR spectral properties with those of an authentic IgG-Fc derived from mammalian cells.

Deletion of plant-specific sugar residues in plant N-glycans by repression of GDP-D-mannose 4,6-dehydratase and ß-1,2-xylosyltransferase genes.

Production of pharmaceutical glycoproteins, such as therapeutic antibodies and cytokines, in plants has many advantages in safety and reduced costs. However, plant-made glycoproteins have N-glycans with plant-specific sugar residues (core ß-1,2-xylose and α-1,3-fucose) and a Lewis a (Le(a)) epitope, Galß(1-3)[Fucα(1-4)]GlcNAc. Because it is likely that these sugar residues and glycan structures are immunogenic, many attempts have been made to delete them. Previously, we reported the simultaneous deletion of the plant-specific core α-1,3-fucose and α-1,4-fucose residues in Le(a) epitopes by repressing the GDP-D-mannose 4,6-dehydratase (GMD) gene, which is associated with GDP-L-fucose biosynthesis, in Nicotiana benthamiana plants (rGMD plants, renamed to ΔGMD plants) (Matsuo and Matsumura, Plant Biotechnol. J., 9, 264-281, 2011). In the present study, we generated a core ß-1,2-xylose residue-repressed transgenic N. benthamiana plant by co-suppression of ß-1,2-xylosyltransferase (ΔXylT plant). By crossing ΔGMD and ΔXylT plants, we successfully generated plants in which plant-specific sugar residues were repressed (ΔGMDΔXylT plants). The proportion of N-glycans with deleted plant-specific sugar residues found in total soluble protein from ΔGMDΔXylT plants increased by 82.41%. Recombinant mouse granulocyte/macrophage-colony stimulating factor (mGM-CSF) and human monoclonal immunoglobulin G (hIgG) harboring N-glycans with deleted plant-specific sugar residues were successfully produced in ΔGMDΔXylT plants. Simultaneous repression of the GMD and XylT genes in N. benthamiana is thus very useful for deleting plant-specific sugar residues.

Development of an efficient agrobacterium-mediated gene targeting system for rice and analysis of rice knockouts lacking granule-bound starch synthase (Waxy) and ß1,2-xylosyltransferase.

We have developed a high-frequency method for Agrobacterium-mediated gene targeting by combining an efficient transformation system using rice suspension-cultured calli and a positive/negative selection system. Compared with the conventional transformation system using calli on solid medium, transformation using suspension-cultured calli resulted in a 5- to 10-fold increase in the number of resistant calli per weight of starting material after positive/negative selection. Homologous recombination occurred in about 1.5% of the positive/negative selected calli. To evaluate the efficacy of our method, we show in this report that knockout rice plants containing either a disrupted Waxy (granule-bound starch synthase) or a disrupted Xyl (ß1,2-xylosyltransferase) gene can be easily obtained by homologous recombination. Study of gene function using homologous recombination in higher plants can now be considered routine work as a direct result of this technical advance.

Small-scale, high-throughput method for plant N-glycan preparation for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis.

A simple, small-scale, and high-throughput method for preparation of plant N-glycans for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is described. This method entailed the extraction of soluble proteins, pepsin digestion, release of N-glycans by glycopeptidase A, and a three-step chromatographic purification process using cation exchange, anion exchange, and graphitized carbon. Homemade minicolumns using commercially available filter unit devices were used for N-glycan purification steps. All purification steps were designed to be easy. Using this method, N-glycans from 10-mg leaf samples of different plant species and only 2 µg of pure horseradish peroxidase were successfully purified.

Deletion of fucose residues in plant N-glycans by repression of the GDP-mannose 4,6-dehydratase gene using virus-induced gene silencing and RNA interference.

Production of pharmaceutical glycoproteins in plants has many advantages in terms of safety and reduced costs. However, plant-produced glycoproteins have N-glycans with plant-specific sugar residues (core ß-1,2-xylose and α-1,3-fucose) and a Lewis a (Le(a) ) epitope, i.e., Galß(1-3)[Fucα(1-4)]GlcNAc. Because these sugar residues and glycan structures seemed to be immunogenic, several attempts have been made to delete them by repressing their respective glycosyltransferase genes. However, until date, such deletions have not been successful in completely eliminating the fucose residues. In this study, we simultaneously reduced the plant-specific core α-1,3-fucose and α-1,4-fucose residues in the Le(a) epitopes by repressing the Guanosine 5'-diphosphate (GDP)-D-mannose 4,6-dehydratase (GMD) gene, which is associated with GDP-L-fucose biosynthesis, in Nicotiana benthamiana plants. Repression of GMD was achieved using virus-induced gene silencing (VIGS) and RNA interference (RNAi). The proportion of fucose-free N-glycans found in total soluble protein from GMD gene-repressed plants increased by 80% and 95% following VIGS and RNAi, respectively, compared to wild-type plants. A small amount of putative galactose substitution in N-glycans from the NbGMD gene-repressed plants was observed, similar to what has been previously reported GMD-knockout Arabidopsis mutant. On the other hand, the recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) with fucose-deleted N-glycans was successfully produced in NbGMD-RNAi transgenic N. benthamiana plants. Thus, repression of the GMD gene is thus very useful for deleting immunogenic total fucose residues and facilitating the production of pharmaceutical glycoproteins in plants.

Development of Cucumber mosaic virus as a vector modifiable for different host species to produce therapeutic proteins.

We have developed Cucumber mosaic virus (CMV) as a plant virus vector especially for production of pharmaceutical proteins. The CMV vector is a vector modifiable for different host plants and does not require further engineering steps. CMV contains three genomic RNA molecules (RNAs 1-3) necessary for infectivity. With this system, instead of creating different vector constructs for each plant we use, we take advantage of the formation of pseudrecombinants between two CMV isolates by simply reassembling a vector construct (RNA 2 base) and an RNA molecule containing the host determinant (mostly RNA 3). In this study, the gene for acidic fibroblast growth factor (aFGF), one of the human cytokines, was cloned under the control of the subgenomic promoter for RNA 4A of the CMV-based vector, C2-H1. Infected Nicotiana benthamiana plants produced aFGF at levels up to 5-8% of the total soluble protein. The tobacco-produced aFGF was purified, and its biological activity was confirmed. Using this system, which provides a versatile and viable strategy for the production of therapeutic proteins in plants, we also demonstrated a high level of aFGF in Glycine max (soybean) and Arabidopsis thaliana.