Proteomic analysis of rice endosperm cells in response to expression of hGM-CSF

The accumulation of significant levels of transgenic products in plant cells is required not only for crop improvement, but also for molecular pharming. However, knowledge about the fate of transgenic products and endogenous proteins in grain cells is lacking. Here, we utilized a quantitative mass spectrometry-based proteomic approach for comparative analysis of expression profiles of transgenic rice endosperm cells in response to expression of a recombinant pharmaceutical protein, human granulocyte-macrophage colony stimulation factor (hGM-CSF). This study provided the first available evidence concerning the fate of exogenous and endogenous proteins in grain cells. Among 1883 identified proteins with a false positive rate of 5%, 103 displayed significant changes (p-value < 0.05) between the transgenic and the wild-type endosperm cells. Notably, endogenous storage proteins and most carbohydrate metabolism-related proteins were down-regulated, while 26S proteasome-related proteins and chaperones were up-regulated in the transgenic rice endosperm. Furthermore, it was observed that expression of hGM-CSF induced endoplasmic reticulum stress and activated the ubiquitin/26S-proteasome pathway, which led to ubiquitination of this foreign gene product in the transgenic rice endosperm.     

Expression and localization of human lysozyme in the endosperm of transgenic rice

In order to understand the characteristics of recombinant protein expression and sublocalization in rice ( Oryza sativa L.) endosperm, we examined the expression level of human lysozyme protein and its subcellular location in transgenic rice seeds driven by rice glutelin and globulin promoters and signal peptides. A time course of human lysozyme expression during endosperm development was analyzed. The results showed that the expression profile of recombinant protein accumulation in endosperm paralleled that of the two storage proteins. Immunofluorescence microscopy revealed that human lysozyme and storage proteins co-localized to type-II protein bodies. Both promoter-signal peptide parings targeted recombinant protein to the protein bodies. In addition, a transgenic line with a higher lysozyme expression level exhibited morphologically different protein bodies with an unbalanced composition of lysozyme and native storage proteins. The high-level expression of recombinant protein distorted the trafficking and sorting of native storage proteins in rice endosperm and affected the expression of native storage protein.     

Molecular pharming in cereal crops

There are many different agricultural expression systems that can be used for the large-scale production of recombinant proteins, but field-grown cereal crops are among the most attractive because recombinant proteins can be targeted to accumulate in the seed, and specifically in the endosperm, which has evolved naturally as a protein storage tissue. Within the developing endosperm, proteins are supplied with molecular chaperones and disulfide isomerases to facilitate folding and assembly, while the mature tissue is desiccated to prevent proteolytic degradation. Proteins expressed in cereal seeds can therefore remain stable for years in ambient conditions. Recent basic research has revealed a surprising diversity of protein targeting mechanisms in the endosperm, which can help to control post-translational modification and accumulation. Applied research and commercial development has seen several pharmaceutical proteins produced in cereals reach late stage preclinical development and the first clinical trials, with a number of companies now dedicated to developing cereal-based production platforms. In this review we discuss the basic science of molecular pharming in cereals, some of the lead product candidates, and challenges that remain to be addressed including the emerging regulatory framework for plant-made pharmaceuticals.     

The endoplasmic reticulum stress induced by highly expressed OsrAAT reduces seed size via pre-mature programmed cell death

The high accumulation of a recombinant protein in rice endosperm causes endoplasmic reticulum (ER) stress and in turn dramatically affects endogenous storage protein expression, protein body morphology and seed phenotype. To elucidate the molecular mechanisms underlying these changes in transgenic rice seeds, we analyzed the expression profiles of endogenous storage proteins, ER stress-related and programmed cell death (PCD)-related genes in transgenic lines with different levels of Oryza sativa recombinant alpha antitrypsin (OsrAAT) expression. The results indicated that OsrAAT expression induced the ER stress and that the strength of the ER stress was dependent on OsrAAT expression levels. It in turn induced upregulation of the expression of the ER stress response genes and downregulation of the expression of the endogenous storage protein genes in rice endosperm. Further experiments showed that the ER stress response upregulated the expression of PCD-related genes to disturb the rice endosperm development and induced pre-mature PCD. As consequence, it resulted in decrease of grain weight and size. The mechanisms for the detriment seed phenotype in transgenic lines with high accumulation of the recombinant protein were elucidated.     

Animal pharming,two decades on

Since its inception 20 years ago, the animal pharming industry has promoted transgenic animals as a cost-effective method of biopharmaceutical production. However, it took until 2006 for the first therapeutic product to gain regulatory approval. This was an important milestone, but scepticism still abounds. Can pharming regain investor confidence, and will society accept transgenic livestock as a production method? There is some cause for optimism, biopharmaceuticals are a large, expanding market and animal pharming has already made considerable strides. A novel production platform has been established, groundbreaking technologies developed, a necessary regulatory framework put in place. Nevertheless, despite cost advantages, pharming has become a niche production method and its long term success may depend on products unique to transgenic animals.     

Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks

Molecular pharming is a cost-effective platform for the production of recombinant proteins in plants. Although the biopharmaceutical industry still relies on a small number of standardized fermentation-based technologies for the production of recombinant proteins there is now a greater awareness of the advantages of molecular pharming particularly in niche markets. Here we discuss some of the technical, economic and regulatory barriers that constrain the clinical development and commercialization of plant-derived pharmaceutical proteins. We also discuss strategies to increase productivity and product quality/homogeneity. The advantages of whole plants should be welcomed by the industry because this will help to reduce the cost of goods and therefore expand the biopharmaceutical market into untapped sectors.     

分子医药农业研究进展

分子医药农业是利用转基因植物为载体,以农业生产的方式规模化生产各种有治疗用途的重组蛋白质及多肽。近20年来,随着植物生物反应器技术的多元化发展以及日趋成熟,植物分子医药农业产业悄然而生。近几年,一些分子医药农业生产的植物源医药产品已实现规模化生产,并进入市场。文中结合国内外最新的研究进展,重点对几种主要植物生物反应器的研究、产品的规模化生产以及产业化进程进行了阐述,以期为我国分子医药农业领域的研究与应用提供参考。     

分子医药农业研究进展

分子医药农业是利用转基因植物为载体,以农业生产的方式规模化生产各种有治疗用途的重组蛋白质及多肽。近20年来,随着植物生物反应器技术的多元化发展以及日趋成熟,植物分子医药农业产业悄然而生。近几年,一些分子医药农业生产的植物源医药产品已实现规模化生产,并进入市场。文中结合国内外最新的研究进展,重点对几种主要植物生物反应器的研究、产品的规模化生产以及产业化进程进行了阐述,以期为我国分子医药农业领域的研究与应用提供参考。     

Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies

This report describes the stable expression of a medically important antibody in the staple cereal crops rice and wheat. We successfully expressed a single-chain Fv antibody (ScFvT84.66) against carcinoembryonic antigen (CEA), a well characterized tumor-associated marker antigen. scFv constructs were engineered for recombinant antibody targeting to the plant cell apoplast and ER. Up to 30 g/g of functional recombinant antibody was detected in the leaves and seeds of wheat and rice. We confirmed that transgenic dry seeds could be stored for at least five months at room temperature, without significant loss of the amount or activity of scFvT84.66. Our results represent the first transition from model plant expression systems, such as tobacco and Arabidopsis, to widely cultivated cereal crops, such as rice and wheat, for expression of an antibody molecule that has already shown efficacy in clinical applications. Thus, we have established that molecular pharming in cereals can be a viable production system for such high-value pharmaceutical macromolecules. Our findings provide a strong foundation for exploiting alternative uses of cereal crops both in industrialized and developing countries.     

Production of human growth hormone in transgenic rice seeds: co-introduction of RNA interference cassette for suppressing the gene expression of endogenous storage proteins

Rice seeds are potentially useful hosts for the production of pharmaceutical proteins. However, low yields of recombinant proteins have been observed in many cases because recombinant proteins compete with endogenous storage proteins. Therefore, we attempt to suppress endogenous seed storage proteins by RNA interference (RNAi) to develop rice seeds as a more efficient protein expression system. In this study, human growth hormone (hGH) was expressed in transgenic rice seeds using an endosperm-specific promoter from a 10 kDa rice prolamin gene. In addition, an RNAi cassette for reduction of endogenous storage protein expressions was inserted into the hGH expression construct. Using this system, the expression levels of 13 kDa prolamin and glutelin were effectively suppressed and hGH polypeptides accumulated to 470 μg/g dry weight at the maximum level in transgenic rice seeds. These results suggest that the suppression of endogenous protein gene expression by RNAi could be of great utility for increasing transgene products.     

Expression of the major mugwort pollen allergen Art v 1 in tobacco plants and cell cultures: problems and perspectives for allergen production in plants

An economic and cheap production of large amounts of recombinant allergenic proteins might become a prerequisite for the common use of microarray-based diagnostic allergy assays which allow a component-specific diagnosis. A molecular pharming strategy was applied to express the major allergen of Artemisia vulgaris pollen, Art v 1, in tobacco plants and tobacco cell cultures. The original Art v 1 with its endogenous signal peptide which directs Art v 1 to the secretory pathway, was expressed in transiently transformed tobacco leaves but was lost in stable transformed tobacco plants during the alternation of generations. Using a light-regulated promoter and “hiding” the recombinant Art v 1 in the ER succeeded in expression of Art v 1 over three generations of tobacco plants and in cell cultures generated from stable transformed plants. However, the amounts of the recombinant allergen were sufficient for analysis but not high enough to allow an economic production. Although molecular pharming has been shown to work well for the production of non-plant therapeutic proteins, it might be less efficient for closely related plant proteins.     

Development of transgenic rice seed accumulating a major Japanese cedar pollen allergen (Cry j 1) structurally disrupted for oral immunotherapy

Rice seed-based edible vaccines expressing T-cell epitope peptides derived from Japanese cedar major pollen allergens have been used to successfully suppress allergen-specific Th2-mediated immunoglobulin E (IgE) responses in mouse experiments. In order to further expand the application of seed-based allergen-specific immunotherapy for controlling Japanese cedar pollinosis, we generated transgenic rice plants that specifically express recombinant Cry j 1 allergens in seeds. Cry j 1 allergens give low specific IgE-binding activity but contain all of the T-cell epitopes. The allergens were expressed directly or as a protein fusion with the major rice storage protein glutelin. Fusion proteins expressed under the control of the strong rice endosperm-specific GluB-1 promoter accumulated in rice endosperm tissue up to 15% of total seed protein. The fusion proteins aggregated with cysteine-rich prolamin and were deposited in endoplasmic reticulum-derived protein body I. The production of transgenic rice expressing structurally disrupted Cry j 1 peptides with low IgE binding activity but spanning the entire Cry j1 region can be used as a universal, safe and effective tolerogen for rice seed-based oral immunotherapy for cedar pollen allergy in humans and other mammals.     

The suppression of the glutelin storage protein gene in transgenic rice seeds results in a higher yield of recombinant protein

Glutelin is a major seed storage protein, accounting for 60?C80?% of the total endosperm protein content in rice. To test whether we could augment the expression of an introduced recombinant protein in rice by suppressing the glutelin gene, we generated transgenic glutelin RNAi (glu RNAi) rice seeds. RNA gel blot analyses confirmed that the endogenous glutelin gene was severely suppressed in these transgenic rice lines. RT-PCR analysis further revealed that all the members of glutelin multigene family were downregulated. Transgenic glu RNAi rice seeds expressing a recombinant red fluorescent protein (RFP) showed stronger fluorescence than seeds transformed with the RFP gene only. Western blot analysis further revealed that the relative accumulation of RFP in glu RNAi seeds was twofold higher than that in the RFP-only transgenic seeds. These results suggest that RNAi targeting of an endogenous storage protein could be of great utility in obtaining higher transgene expression in genetically engineered rice and other plant lines.     

Improvement of Human lysozyme Expression in Transgenic Rice Grain by Combining Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) <Emphasis Type="Italic">puroindoline b</Emphasis> and Rice <Emphasis Type="Italic">(Oryza sativa)</Emphasis> Gt1 Promoters and Signal Peptides

Heterologous protein expression levels in transgenic plants are of critical importance in the production of plant-made pharmaceuticals (PMPs). We studied a puroindoline b promoter and signal peptide (Tapur) driving human lysozyme expression in rice endosperm. The results demonstrated that human lysozyme expressed under the control of the Tapur cassette is seed-specific, readily extractable, active, and properly processed. Immuno-electron microscopy indicated that lysozyme expressed from this cassette is localized in protein bodies I and II in rice endosperm cells, demonstrating that this non-storage promoter and signal peptide can be used for targeting human lysozyme to rice protein bodies. We successfully employed a strategy to improve the expression of human lysozyme in transgenic rice grain by combining the Tapur cassette with our well established Gt1 expression system. The results demonstrated that when the two expression cassettes were combined, the expression level of human lysozyme increased from 5.24 ± 0.34 mg⁻¹ g flour for the best single cassette line to 9.24 ± 0.06 mg⁻¹ g flour in the best double cassette line, indicating an additive effect on expression of human lysozyme in rice grain.     

Expression of Cholera Toxin B Subunit in Transgenic Rice Endosperm

The synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized codon usage of plant genes, was introduced into a plant expression vector and expressed under the control of the Bx17 HMW (high molecular weight) wheat endosperm-specific promoter containing an intron of the rice act1. The recombinant vector was transformed into rice plants using a biolistic-mediated transformation method. Stable integration of the synthetic CTB gene into the chromosomal DNA was confirmed by PCR amplification analysis. A high level of CTB (2.1% of total soluble protein) was expressed in the endosperm tissue of the transgenic rice plants. The synthetic CTB produced only in the rice endosperm demonstrated strong affinity for G_M1-ganglioside, thereby suggesting that the CTB subunits formed an active pentamer. The successful expression of CTB genes in transgenic plants makes it a powerful tool for the development of a plant-derived edible vaccine.