Biofactories: producció de proteïnes recombinants en plantes

Fins al començament del segle xxi, les plantes havien estat la font de remeis per a malalties en humans. Després, els extractes vegetals van ser substituïts per productes produïts a escala industrial per síntesi química. Durant les darreres dècades del segle xx, va aparèixer una alternativa a la síntesi química gràcies al desenvolupament de l'enginyeria genètica en bacteris, llevats i cèl·lules animals per a la producció de proteïnes recombinants. Amb el desenvolupament recent de les metodologies de transformació vegetal, les plantes ofereixen un sistema alternatiu al bacterià o al de cultiu de cèl·lules animals, per a la producció de proteïnes recombinants a baix cost i segur per a la salut humana. En aquest article il·lustrem els avantatges i desavantatges de diferents sistemes vegetals (transformació estable i transitòria), les seves limitacions i reptes. També exposem les millores que actualment s'estan duent a terme, emprant espècies diferents de plantes i sistemes diferents d'expressió per tal de consolidar aquestes factories per a la producció de proteïnes terapèutiques senzilles o molt complexes.Plants were the main source for human drugs until the beginning of the nineteenth century when plant-made pharmaceuticals were partly supplanted by drugs produced by the industrial methods of chemical synthesis. During the last decades of twentieth century, genetic engineering has offered an alternative to chemical synthesis using bacteria, yeast and animal cells as a factories for the production of recombinant proteins. With the recent development of plant-based recombinant protein production systems, plants offer a safe and extremely cost effective alternative to microbial and mammalian cell cultures. Here, we evaluate the advantages and disadvantages of different plant expression systems (stable nuclear, or transient transformations) and their current limitations or challenges. We also illustrate that current improvements in plant expression systems and plant hosts are making them suitable as alternative factories for the production of either simple or highly complex recombinant proteins

Differential Protein Accumulation in Banana Fruit during Ripening

6 pages, 4 figures.-- PMID: 16668607 [PubMed].-- PMCID: PMC1080163.Banana (Musa acuminata, cv Dwarf Cavendish) proteins were extracted from pulp tissue at different stages of ripening and analyzed by two-dimensional electrophoresis. The results provide evidence of differential protein accumulation during ripening. Two sets of polypeptides have been detected that increase substantially in ripe fruit. These polypeptides were characterized as glycoproteins by western blotting and concanavalin A binding assays. Antibodies againts tomato polygalacturonase cross-react with one of these sets of proteins.This work was supported by grant ALI-88-0138 from Comisión Interministerial de Ciencia y Technologia, and grant AR/88 from the Comissió Interdepartamental de Recerca e Innovació Tocnologica Generalitat de Catalunya. E.D.-P. is a recipient of a fellowship from the Ministerio Educatión y Ciencia.Peer reviewe

The expression pattern of the tonoplast intrinsic protein γ-TIP in Arabidopsis thaliana Is correlated with cell enlargement

The vacuolar membrane (tonoplast) contains an abundant intrinsic protein with six membrane-spanning domains that is encoded by a small gene family. Different isoforms of tonoplast intrinsic protein (TIP) are expressed in different tissues or as a result of specific signals. Using promoter-beta-glucuronidase (GUS) fusions and in situ hybridization, we have examined the expression of gamma-TIP in Arabidopsis thaliana. GUS staining of plants transformed with promoter-GUS fusions showed that gamma-TIP gene expression is high in recently formed tissues of young roots. In the shoot, gamma-TIP gene expression was highest in the vascular bundles of stems and petioles, as well as in the stipules and in the receptacle of the flower. No GUS activity was detected in root or shoot meristems or in older tissues, suggesting temporal control of gamma-TIP gene expression associated with cell elongation and/or differentiation. In situ hybridization carried out with whole seedlings confirmed that in root tips, gamma-TIP mRNA was present only in the zone of cell elongation just behind the apical meristem. In seedling shoots, mRNA abundance was also found to be correlated with cell expansion. These results indicate that gamma-TIP may be expressed primarily at the time when the large central vacuoles are being formed during cell enlargement.Supported by a grant from the United States Department of Agriculture (to M.J.C.), a European Molecular Biology Organization fellowship (to H.H.), and a fellowship from the Provincial Government of Catalonia (to D.L.).Peer reviewe

Proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves

<p>Abstract</p> <p>Background</p> <p>The N-terminal proline-rich domain (Zera) of the maize storage protein γ-zein, is able to induce the formation of endoplasmic reticulum (ER)-derived protein bodies (PBs) when fused to proteins of interest. This encapsulation enables a recombinant fused protein to escape from degradation and facilitates its recovery from plant biomass by gradient purification. The aim of the present work was to evaluate if induced PBs encapsulate additional proteins jointly with the recombinant protein. The exhaustive analysis of protein composition of PBs is expected to facilitate a better understanding of PB formation and the optimization of recombinant protein purification approaches from these organelles.</p> <p>Results</p> <p>We analysed the proteome of PBs induced in <it>Nicotiana benthamiana </it>leaves by transient transformation with Zera fused to a fluorescent marker protein (DsRed). Intact PBs with their surrounding ER-membrane were isolated on iodixanol based density gradients and their integrity verified by confocal and electron microscopy. SDS-PAGE analysis of isolated PBs showed that Zera-DsRed accounted for around 85% of PB proteins in term of abundance. Differential extraction of PBs was performed for in-depth analysis of their proteome and structure. Besides Zera-DsRed, 195 additional proteins were identified including a broad range of proteins resident or trafficking through the ER and recruited within the Zera-DsRed polymer.</p> <p>Conclusions</p> <p>This study indicates that Zera-protein fusion is still the major protein component of the new formed organelle in tobacco leaves. The analysis also reveals the presence of an unexpected diversity of proteins in PBs derived from both the insoluble Zera-DsRed polymer formation, including ER-resident and secretory proteins, and a secretory stress response induced most likely by the recombinant protein overloading. Knowledge of PBs protein composition is likely to be useful to optimize downstream purification of recombinant proteins in molecular farming applications.</p

Artificially-induced organelles are optimal targets for optical trapping experiments in living cells

Optical trapping supplies information on the structural, kinetic or rheological properties of inner constituents of the cell. However, the application of significant forces to intracellular objects is notoriously difficult due to a combination of factors, such as the small difference between the refractive indices of the target structures and the cytoplasm. Here we discuss the possibility of artificially inducing the formation of spherical organelles in the endoplasmic reticulum, which would contain densely packed engineered proteins, to be used as optimized targets for optical trapping experiments. The high index of refraction and large size of our organelles provide a firm grip for optical trapping and thereby allow us to exert large forces easily within safe irradiation limits. This has clear advantages over alternative probes, such as subcellular organelles or internalized synthetic beads

The Expression of a Xylanase Targeted to ER-Protein Bodies Provides a Simple Strategy to Produce Active Insoluble Enzyme Polymers in Tobacco Plants

Background Xylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs). Methodology/Principal Findings Tobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase. Conclusion/Significance In the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes.This work was mainly supported by ERA Biotech (www.erabiotech.com). Additional support was supplied by grant SGR 2009/703 funded by the Generalitat de Catalunya (www10.gencat.net) and grants CDS2007/00036 of Consolider Ingenio program and TRA 2009/0124 of TRACE program funded by Ministerio de Ciencia e Inovación (MICINN, www.micinn.es). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

The Expression Pattern of the Tonoplast Intrinsic Protein γ-TIP in \u3cem\u3eArabidopsis thaliana\u3c/em\u3e Is Correlated With Cell Enlargement

The vacuolar membrane (tonoplast) contains an abundant intrinsic protein with six membrane-spanning domains that is encoded by a small gene family. Different isoforms of tonoplast intrinsic protein (TIP) are expressed in different tissues or as a result of specific signals. Using promoter-β-glucuronidase (GUS) fusions and in situ hybridization, we have examined the expression of γ-TIP in Arabidopsis thaliana. GUS staining of plants transformed with promoter-GUS fusions showed that γ-TIP gene expression is high in recently formed tissues of young roots. In the shoot, γ-TIP gene expression was highest in the vascular bundles of stems and petioles, as well as in the stipules and in the receptacle of the flower. No GUS activity was detected in root or shoot meristems or in older tissues, suggesting temporal control of γ-TIP gene expression associated with cell elongation and/or differentiation. In situ hybridization carried out with whole seedlings confirmed that in root tips, γ-TIP mRNA was present only in the zone of cell elongation just behind the apical meristem. In seedling shoots, mRNA abundance was also found to be correlated with cell expansion. These results indicate that γ-TIP may be expressed primarily at the time when the large central vacuoles are being formed during cell enlargement

Molecular farming: Plant made biopharmaceuticals

[CA] Fins al començament del segle xxi, les plantes havien estat la font de remeis per a malalties en humans. Després, els extractes vegetals van ser substituïts per productes produïts a escala industrial per síntesi química. Durant les darreres dècades del segle xx, va aparèixer una alternativa a la síntesi química gràcies al desenvolupament de l’enginyeria genètica en bacteris, llevats i cèl·lules animals per a la producció de proteïnes recombinants. Amb el desenvolupament recent de les metodologies de transformació vegetal, les plantes ofereixen un sistema alternatiu al bacterià o al de cultiu de cèl·lules animals, per a la producció de proteïnes recombinants a baix cost i segur per a la salut humana. En aquest article il·lustrem els avantatges i desavantatges de diferents sistemes vegetals (transformació estable i transitòria), les seves limitacions i reptes. També exposem les millores que actualment s’estan duent a terme, emprant espècies diferents de plantes i sistemes diferents d’expressió per tal de consolidar aquestes factories per a la producció de proteïnes terapèutiques senzilles o molt complexes.[EN] Plants were the main source for human drugs until the beginning of the nineteenth century when plant-made pharmaceuticals were partly supplanted by drugs produced by the industrial methods of chemical synthesis. During the last decades of twentieth century, genetic engineering has offered an alternative to chemical synthesis using bacteria, yeast and animal cells as a factories for the production of recombinant proteins. With the recent development of plant-based recombinant protein production systems, plants offer a safe and extremely cost effective alternative to microbial and mammalian cell cultures. Here, we evaluate the advantages and disadvantages of different plant expression systems (stable nuclear, or transient transformations) and their current limitations or challenges. We also illustrate that current improvements in plant expression systems and plant hosts are making them suitable as alternative factories for the production of either simple or highly complex recombinant proteins.Peer reviewe