25 research outputs found
Non-food/feed seeds as biofactories for the high-yield production of recombinant pharmaceuticals
No full textWe describe an attractive cloning system for the seed-specific expression of recombinant proteins using three non-food/feed crops. A vector designed for direct subcloning by Gateway ® recombination was developed and tested in Arabidopsis, tobacco and petunia plants for the production of a chimeric form (GAD67/65) of the 65kDa isoform of glutamic acid decarboxylase (GAD65). GAD65 is one of the major human autoantigens involved in type 1 diabetes (T1D). The murine anti-inflammatory cytokine interleukin-10 (IL-10) was expressed with the described system in Arabidopsis and tobacco, whereas proinsulin, another T1D major autoantigen, was expressed in Arabidopsis. The cost-effective production of these proteins in plants could allow the development of T1D prevention strategies based on the induction of immunological tolerance. The best yields were achieved in Arabidopsis seeds, where GAD67/65 reached 7.7% of total soluble protein (TSP), the highest levels ever reported for this protein in plants. IL-10 and proinsulin reached 0.70% and 0.007% of TSP, respectively, consistent with levels previously reported in other plants or tissues. This versatile cloning vector could be suitable for the high-throughput evaluation of expression levels and stability of many valuable and difficult to produce proteins. © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd
Non-food/feed seeds as biofactories for the high-yield production of recombinant pharmaceuticals.
No full textWe describe an attractive cloning system for the seed-specific expression of recombinant proteins using three non-food/feed crops. A vector designed for direct subcloning by Gateway(®) recombination was developed and tested in Arabidopsis, tobacco and petunia plants for the production of a chimeric form (GAD67/65) of the 65 kDa isoform of glutamic acid decarboxylase (GAD65). GAD65 is one of the major human autoantigens involved in type 1 diabetes (T1D). The murine anti-inflammatory cytokine interleukin-10 (IL-10) was expressed with the described system in Arabidopsis and tobacco, whereas proinsulin, another T1D major autoantigen, was expressed in Arabidopsis. The cost-effective production of these proteins in plants could allow the development of T1D prevention strategies based on the induction of immunological tolerance. The best yields were achieved in Arabidopsis seeds, where GAD67/65 reached 7.7% of total soluble protein (TSP), the highest levels ever reported for this protein in plants. IL-10 and proinsulin reached 0.70% and 0.007% of TSP, respectively, consistent with levels previously reported in other plants or tissues. This versatile cloning vector could be suitable for the high-throughput evaluation of expression levels and stability of many valuable and difficult to produce proteins
Russell-like bodies sequestering recombinant proteins
No full textMost molecular farming applications have in common the need for high yields of recombinant protein which can be achieved by increasing expression and ensuring the stable accumulation of functional proteins in the most appropriate subcellular locations. Targeting strategies can be used to enhance the accumulation of a protein, and to control posttranslational modifications such as glycosylation. The success of such strategies is variable and appears to depend on the plant species and tissue, but also on the protein under study. In some cases, targeting strategies have to be adapted to specialized tissues to achieve the desired effect, e.g. the deposition in native storage organelles such as protein bodies or protein storage vacuoles of seeds. In other cases, aberrant localization can be attributed to inherent properties of the protein. For example, the unintended accumulation in ER-derived compartments may arise from covalent interactions with endogenous ER-resident proteins. Thus the partial localization of a recombinant antibody in the periphery of prolamin storage organelles was observed, most likely due an interaction between the heavy chain of the antibody and gamma-zein, an endogenous storage protein of maize.
Even in the absence of endogenous ER-derived storage organelles, recombinant proteins are occasionally sequestered in ER-derived bodies. In Arabidopsis seeds we have identified an insoluble fraction of recombinant murine interleukin-10 and localized this fraction within ER-derived protein accretions. Electron tomography demonstrates that they are detached from the ER and appear very similar to Russell bodies, which occur in connection with human ER storage diseases. Another example was observed in antibody- producing tobacco leaves, where an insoluble proportion of the recombinant protein was detected in ER- derived vesicular structures. We speculate that the content of these ER-bodies corresponds to a transport- incompetent fraction of the recombinant protein, and that, similar to mammalian plasma cells, plant cells are able to form Russell bodies as a self-protection mechanism to avoid blockage of the secretory pathway
Proteome of tolerance fine-tuning in the human pathogen black yeast Exophiala dermatitidis
No full textAbstractThe black yeast Exophiala dermatitidis is a worldwide distributed agent of primary and secondary diseases in both immunocompromised and healthy humans, with a high prevalence in human-made environments. Since thermo-tolerance has a crucial role in the fungus persistence in man-dominated habitat and in its pathogenicity, three incubation temperatures (37, 45, 1°C) and two time spans (1h, 1week) were selected to simulate different environmental conditions and to investigate the effect of temperature on the proteome of E. dermatitidis CBS 525.76. Using a novel protocol for protein extraction from black yeasts, 2-D DIGE could be applied for characterization of changes in total protein spot abundance among the experimental conditions. A total of 32 variable proteins were identified by mass spectrometry. Data about protein functions, localization and pathways were also obtained. A typical stress response under non-optimal temperature could not be observed at the proteome level, whereas a reduction of the metabolic activity, mostly concerning processes as the general carbon metabolism, was detected after exposure to cold. These results suggest that a fine protein modulation takes place following temperature treatment and a repertoire of stable protein might be at the base of E. dermatitidis adaptation to altered growth conditions.SignificanceE. dermatitidis is a pathogenic black yeast causing neurotropic infections, systemic and subcutaneous disease in a wide range of hosts, including humans. The discovery of the fungus high prevalence in man-made habitats, including sauna facilities, drinking water and dishwashers, generated concern and raised questions about the infection route. In the present work — which is the first contribution on E. dermatitidis proteome — the effect of different temperature conditions on the fungus protein pattern have been analyzed by using a gel-based approach and the temperature responsive proteins have been identified.The absence of a typical stress response following the exposure to non-optimal temperature was detected at the proteome level, along with a general reduction of the metabolic activity after exposure to cold. These results suggest that a very fine regulation of the protein expression as well as adaptations involving a basic set of stable proteins may be at the base of E. dermatitidis enormous ecological plasticity, which plays a role in the fungus distribution, also enabling the transition from natural to human habitat and to the human host
Rice endosperm produces an underglycosylated and potent form of the HIV-neutralizing monoclonal antibody 2G12.
No full textPlant species and organ influence the structure and subcellular localization of recombinant glycoproteins
No full textS.105-117Many plant-based systems have been developed as bioreactors to produce recombinant proteins. The choice of system for large-scale production depends on its intrinsic expression efficiency and its propensity for scale-up, post-harvest storage and downstream processing. Factors that must be considered include the anticipated production scale, the value and intended use of the product, the geographical production area, the proximity of processing facilities, intellectual property, safety and economics. It is also necessary to consider whether different species and organs affect the subcellular trafficking, structure and qualitative properties of recombinant proteins. In this article we discuss the subcellular localization and N-glycosylation of two commercially-relevant recombinant glycoproteins (Aspergillus niger phytase and anti-HIV antibody 2G12) produced in different plant species and organs. We augment existing data with novel results based on the expression of the sa me recombinant proteins in Arabidopsis and tobacco seeds, focusing on similarities and subtle differences in N-glycosylation that often reflect the subcellular trafficking route and final destination, as well as differences generated by unique enzyme activities in different species and tissues. We discuss the potential consequences of such modifications on the stability and activity of the recombinant glycoproteins.83Nr.1-
Unexpected deposition patterns of recombinant proteins in post-endoplasmic reticulum compartments of wheat endosperm
No full textS.3457-3466Protein transport within cereal endosperm cells is complicated by the abundance of endoplasmic reticulum (ER)-derived and vacuolar protein bodies. For wheat storage proteins, two major transport routes run from the ER to the vacuole, one bypassing and one passing through the Golgi. Proteins traveling along each route converge at the vacuole and form aggregates. To determine the impact of this trafficking system on the fate of recombinant proteins expressed in wheat endosperm, we used confocal and electron microscopy to investigate the fate of three recombinant proteins containing different targeting information. KDEL-tagged recombinant human serum albumin, which is retrieved to the ER lumen in leaf cells, was deposited in prolamin aggregates within the vacuole of endosperm cells, most likely following the bulk of endogenous glutenins. Recombinant fungal phytase, a glycoprotein designed for secretion, was delivered to the same compartment, with no trace of the molecule in the apoplast. Glycan analysis revealed that this protein had passed through the Golgi. The localization of human serum albumin and phytase was compared to that of recombinant legumin, which contains structural targeting information directing it to the vacuole. Uniquely, legumin accumulated in the globulin inclusion bodies at the periphery of the prolamin bodies, suggesting a different mode of transport and/or aggregation. Our results demonstrate that recombinant proteins are deposited in an unexpected pattern within wheat endosperm cells, probably because of the unique storage properties of this tissue. Our data also confirm that recombinant proteins are invaluable tools for the analysis of protein trafficking in cereals.136Nr.
From recombinant proteins to plant-made-pharmaceuticals
No full textS.S17The plant-based production of biopharmaceuticals has attracted interest because plants, compared to traditional platforms for the production of recombinant proteins, are inexpensive, highly scalable and safe. The number of products in development is increasing as tools and strategies have been developed to accumulate recombinant proteins with specific glycan modifications in various plant species and organs. Different platforms have been established including transient expression systems, contained systems based on cultured plant cells, and stable transgenic plants accumulating recombinant proteins in leaves, seeds, fruits or tubers/roots. Plant systems appear particularly suitable for the production of antibodies that are required in large amounts. Antibody 2G12 for example is one of a small number of human IgG monoclonal antibodies exhibiting potent and broad HIV-1-neutralizing activity in vitro, and the ability to prevent HIV-1 infection in animal models. We have expressed this antibody in tobacco and maize, which could facilitate inexpensive, large-scale production. The specific antigen-binding function of the antibody purified from both plant species was verified by surface plasmon resonance analysis, and in vitro cell assays demonstrated that the HIV-neutralizing properties of the plant-produced antibody were equivalent to or better than those of its CHO-derived counterpart. Clinical studies with plant-derived antibody are being initiated.22Suppl.
Enhancing quality and yield of recombinant secretory IgA antibodies in Nicotiana benthamiana by endoplasmic reticulum engineering.
No full textThe production of complex multimeric secretory immunoglobulins (SIgA) in Nicotiana benthamiana leaves is challenging, with significant reductions in complete protein assembly and consequently yield, being the most important difficulties. Expanding the physical dimensions of the ER to mimic professional antibody-secreting cells can help to increase yields and promote protein folding and assembly. Here, we expanded the ER in N. benthamiana leaves by targeting the enzyme CTP:phosphocholine cytidylyltransferase (CCT), which catalyses the rate-limiting step in the synthesis of the key membrane component phosphatidylcholine (PC). We used CRISPR/Cas to perform site-directed mutagenesis of each of the three endogenous CCT genes in N. benthamiana by introducing frame-shifting indels to remove the auto-inhibitory C-terminal domains. We generated stable homozygous lines of N. benthamiana containing different combinations of the edited genes, including plants where all three isofunctional CCT homologues were modified. Changes in ER morphology in the mutant plants were confirmed by in vivo confocal imaging and substantially increased the yields of two fully assembled SIgAs by prolonging the ER residence time and boosting chaperone accumulation. Through a combination of ER engineering with chaperone overexpression, we increased the yields of fully assembled SIgA by an order of magnitude, reaching almost 1 g/kg fresh leaf weight. This strategy removes a major roadblock to producing SIgA and will likely facilitate the production of other complex multimeric biopharmaceutical proteins in plants
