74 research outputs found
Enhanced GAD65 production in plants using the MagnICON transient expression system: Optimization of upstream production and downstream processing
No full textPlants have emerged as competitive production platforms for pharmaceutical proteins that are required in large quantities. One example is the 65-kDa isoform of human glutamic acid decarboxylase (GAD65), a major autoimmune diabetes autoantigen that has been developed as a vaccine candidate for the primary prevention of diabetes. The expression of GAD65 in plants has been optimized but large-scale purification is hampered by its tendency to associate with membranes. We investigated the potential for large-scale downstream processing by evaluating different combinations of plant-based expression systems and engineered forms of GAD65 in terms of yield, subcellular localization and solubility in detergent-free buffer. We found that a modified version of GAD65 lacking the first 87 amino acids accumulates to high levels in the cytosol and can be extracted in detergent-free buffer. The highest yields of this variant protein were achieved using the MagnICON transient expression system. This combination of truncated GAD65 and the MagnICON system dramatically boosts the production of the recombinant protein and helps to optimize downstream processing for the establishment of a sustainable plant-based production platform for an autoimmune diabetes vaccine candidate
Utilizing storage organelles for stable accumulation of recombinant proteins and biopharmaceuticals
No full textUsing storage organelles for the accumulation and encapsulation of recombinant proteins
No full textPlants have been used to produce many diverse and valuable recombinant proteins, including subunit vaccines, antibodies and antibody fragments, hormones, blood products, cytokines, and enzymes. Different plant species and platforms have been explored as production hosts, each with unique properties in terms of the gene transfer method, production time, environmental containment, scalability, downstream processing strategy, protein folding and accumulation, and overall costs. Seed-based systems have many advantages because they exploit the natural storage properties of seeds, which facilitate batch processing and distribution. Seeds possess specialized storage organelles that may be used to accumulate recombinant proteins, offering stability both in planta and after harvest in the final preparation/formulation. The post-harvest stabilizing effect of seeds allows recombinant subunit vaccines and antibodies to be delivered via the mucosal route because they are better able to withstand this harsh microenvironment when protected by the plant matrix. Native storage organelles such as starch granules and protein bodies offer this protective effect, but protein storage organelles can also be induced ectopically in vegetative tissues. In this paper, we discuss the technical capabilities of storage organelle-based expression platforms and their potential applications
Progressive Aggregation of 16 kDa Gamma-Zein during Seed Maturation in Transgenic Arabidopsis thaliana
No full textProlamins constitute a unique class of seed storage proteins, present only in grasses. In the lumen of the endoplasmic reticulum (ER), prolamins form large, insoluble heteropolymers termed protein bodies (PB). In transgenic Arabidopsis (Arabidopsis thaliana) leaves, the major maize (Zea mays) prolamin, 27 kDa γ-zein (27γz), assembles into insoluble disulfide-linked polymers, as in maize endosperm, forming homotypic PB. The 16 kDa γ-zein (16γz), evolved from 27γz, instead forms disulfide-bonded dispersed electron-dense threads that enlarge the ER lumen without assembling into PB. We have investigated whether the peculiar features of 16γz are also maintained during transgenic seed development. We show that 16γz progressively changes its electron microscopy appearance during transgenic Arabidopsis embryo maturation, from dispersed threads to PB-like, compact structures. In mature seeds, 16γz and 27γz PBs appear very similar. However, when mature embryos are treated with a reducing agent, 27γz is fully solubilized, as expected, whereas 16γz remains largely insoluble also in reducing conditions and drives insolubilization of the ER chaperone BiP. These results indicate that 16γz expressed in the absence of the other zein partners forms aggregates in a storage tissue, strongly supporting the view that 16γz behaves as the unassembled subunit of a large heteropolymer, the PB, and could have evolved successfully only following the emergence of the much more structurally self-sufficient 27γz
Protein functional analysis data in support of comparative proteomics of the pathogenic black yeast Exophiala dermatitidis under different temperature conditions
No full textAbstractIn the current study a comparative proteomic approach was used to investigate the response of the human pathogen black yeast Exophiala dermatitidis toward temperature treatment. Protein functional analysis – based on cellular process GO terms – was performed on the 32 temperature-responsive identified proteins. The bioinformatics analyses and data presented here provided novel insights into the cellular pathways at the base of the fungus temperature tolerance. A detailed analysis and interpretation of the data can be found in “Proteome of tolerance fine-tuning in the human pathogen black yeast Exophiala dermatitidis” by Tesei et al. (2015) [1]
Heat-stable phytases in transgenic wheat (Triticum aestivum l.): Deposition pattern, thermostability, and phytate hydrolysis
No full textPlant species and organ influence the structure and subcellular localization of recombinant glycoproteins
No full text
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
- …
