Optimierung der Photosynthese von C3-Pflanzen : C 4 -ähnlicher Zyklus und chloroplastidärer Bypass

Most agronomic traits are polygenic in nature and rely on complex metabolic and regulatory pathways. Genetic modifications of these traits or introduction of new pathways require the transfer of multiple genes into the plant genome. Most of the existing methods like retransformation, cotransformation and crossing suffer from certain deficiencies like high expenditure of time or the integration of multiple copies (Dafny-Yelin und Tzfira, 2007; Naqvi et al., 2010). The transfer of the genes as a single multigene construct offers considerable advantages. Up to now there are only few efficient methods for assembly and subsequent transformation of multigene constructs into plants. In the context of this work a Gateway-based method could be established, which enables fusion of multiple fragments. The system consists of a Gateway-compatible destination vector and two special entry vectors with attR cassettes, which are flanked by incompatible attL sequences. By alternate use of the two entry vectors multiple transgenes can be recombined into the destination vector. Multigene constructs with 7 gene expression cassettes could be transferred successfully into tobacco by agrobacterium-mediated transformation. By biolistic transformation a multigene construct with 5 gene expression cassettes could be transferred into rice. Due to the bifunctionality of RUBISCO, the key enzyme of photosynthesis, besides carboxylation also oxygenation of ribulose-1,5-bisphosphat is catalyzed in the chloroplasts of higher plants (Bowes et al., 1971). Glycolate formed by this process is toxic and unusable for the plant (Zelitch et al., 2008) and have to be metabolized in the photorespiration. The conversion of glycolate not only consumes ATP and reduction equivalents, it also leads to loss of 25% carbon fixed in this metabolite. For C3 plants photorespiration means a significant reduction of photosynthesis efficiency. On the contrary C4 plants can considerably reduce the oxygenase activity of RUBISCO due to their CO2 concentration mechanism. By use of the MultiRound Gateway technology tobacco plants could be generated, which expressed the heterologous enzymes for a single cell C4 cycle following the example of Hydrilla verticillata. Besides a NADP-ME type C4 cylce, a NAD-ME type and a PCK type C4 cycle were constructed. However, there was no evidence for a CO2 concentration inside the chloroplasts. The heterologous expression of both malic enzymes (NADP-HvMe and NAD-EcMe) strongly affects plant growth. The higher the ME activities were, the slower was the plant growth. All types had in common a higher nitrogen content, which resulted in a lower C/N ratio. Also by use of the MultiRound Gateway technology rice plants with a chloroplastic photorespiratory bypass could be generated. The bypass relies on the catabolic glycolate pathway from E. coli and converts the glycolate inside the chloroplasts. This is intended to increase the CO2 concentration in the vicinity of RUBISCO and thereby suppress photorespiration. In Arabidopsis this pathway led to promising results (Kebeish et al., 2007). The expression of all genes required for this pathway (TSR, GCL, glcD, glcE, glcF) could be verified on the RNA level. To sum up, it can be said that the MultiRound Gateway technology is well suited for the assembly of multigene constructs and the constructed destination vector can be transferred into plants by agrobacterium-mediated transformation as well as biolistic transformation. The generated plants can serve as starting basis for further attempts to establish a single cell C4 cycle. A better balanced relation of enzymatic activities may avoid the negative effects on plant growth caused by high ME activities

Tobacco BY-2 cell-free lysate : an alternative and highly-productive plant-based in vitro translation system

Background: Cell-free protein synthesis is a rapid and efficient method for the production of recombinant proteins. Usage of prokaryotic cell-free extracts often leads to non-functional proteins. Eukaryotic counterparts such as wheat germ extract (WGE) and rabbit reticulocyte lysate (RLL) may improve solubility and promote the correct folding of eukaryotic multi-domain proteins that are difficult to express in bacteria. However, the preparation of WGEs is complex and time-consuming, whereas RLLs suffer from low yields. Here we report the development of a novel cell-free system based on tobacco Bright Yellow 2 (BY-2) cells harvested in the exponential growth phase. Results: The highly-productive BY-2 lysate (BYL) can be prepared quickly within 4-5 h, compared to 4-5 d for WGE. The efficiency of the BYL was tested using three model proteins: enhanced yellow fluorescent protein (eYFP) and two versions of luciferase. The added mRNA was optimized by testing different 5' and 3' untranslated regions (UTRs). The protein yield in batch and dialysis reactions using BYL was much higher than that of a commercial Promega WGE preparation, achieving a maximum yield of 80 mu g/mL of eYFP and 100 mu g/mL of luciferase, compared to only 45 mu g/mL of eYFP and 35 mu g/mL of luciferase in WGEs. In dialysis reactions, the BYL yielded about 400 mu g/mL eYFP, representing up to 50% more of the target protein than the Promega WGE, and equivalent to the amount using 5Prime WGE system. Conclusions: Due to the high yield and the short preparation time the BYL represents a remarkable improvement over current eukaryotic cell-free systems

Cell-free protein synthesis system

The technology provided herein relates to novel methods and systems for cell-free protein synthesis, in particular to cell-free protein synthesis systems suitable for protein synthesis before and after freezing for a short- or long-term storage, wherein said system comprises a cryoprotectant

Promoter construct for cell-free protein synthesis

The technology provided herein relates to novel promoter constructs, method and systems for an increase in the target protein yield and/or allowing the use of lower template concentration in the cell-free protein synthesis systems

Critical Analysis of the Commercial Potential of Plants for the Production of Recombinant Proteins

Over the last three decades, the expression of recombinant proteins in plants and plant cells has been promoted as an alternative cost-effective production platform. However, the market is still dominated by prokaryotic and mammalian expression systems, the former offering high production capacity at a low cost, and the latter favored for the production of complex biopharmaceutical products. Although plant systems are now gaining widespread acceptance as a platform for the larger-scale production of recombinant proteins, there is still resistance to commercial uptake. This partly reflects the relatively low yields achieved in plants, as well as inconsistent product quality and difficulties with larger-scale downstream processing. Furthermore, there are only a few cases in which plants have demonstrated economic advantages compared to established and approved commercial processes, so industry is reluctant to switch to plant-based production. Nevertheless, some plant-derived proteins for research or cosmetic/pharmaceutical applications have reached the market, showing that plants can excel as a competitive production platform in some niche areas. Here, we discuss the strengths of plant expression systems for specific applications, but mainly address the bottlenecks that must be overcome before plants can compete with conventional systems, enabling the future commercial utilization of plants for the production of valuable proteins.Additional funding: Projects Phe-free 2 (FZK 031A587B) and Cell-Free Biosynthesis (FKZ 0315942) funded by the Federal Ministry of Education and Research (BMBF

Combination of two epitope identification techniques enables the rational design of soy allergen Gly m 4 mutants

Detailed IgE-binding epitope analysis is a key requirement for the understanding and development of diagnostic and therapeutic agents to address food allergies. An IgE-specific linear peptide microarray with random phage peptide display for the high-resolution mapping of IgE-binding epitopes of the major soybean allergen Gly m 4, which is a homologue to the birch pollen allergen Bet v 1 is combined. Three epitopes are identified and mapped to a resolution of four key amino acids, allowing the rational design and the production of three Gly m 4 mutants with the aim to abolish or reduce the binding of epitope-specific IgE. In ELISA, the binding of the mutant allergens to polyclonal rabbit-anti Gly m 4 serum as well as IgE purified from Gly m 4-reactive soybean allergy patient sera is reduced by up to 63% compared to the wild-type allergen. Basophil stimulation experiments using RBL-SX38 cells loaded with patient IgE are showed a decreased stimulation from 25% for the wild-type Gly m 4 to 13% for one mutant. The presented approach demonstrates the feasibility of precise mapping of allergy-related IgE-binding epitopes, allowing the rational design of less allergenic mutants as potential therapeutic agent

Combined 15N-Labeling and TandemMOAC Quantifies Phosphorylation of MAP Kinase Substrates Downstream of MKK7 in Arabidopsis

Reversible protein phosphorylation is a widespread posttranslational modification that plays a key role in eukaryotic signal transduction. Due to the dynamics of protein abundance, low stoichiometry and transient nature of protein phosphorylation, the detection and accurate quantification of substrate phosphorylation by protein kinases remains a challenge in phosphoproteome research. Here, we combine tandem metal-oxide affinity chromatography (tandemMOAC) with stable isotope 15N metabolic labeling for the measurement and accurate quantification of low abundant, transiently phosphorylated peptides by mass spectrometry. Since tandemMOAC is not biased toward the enrichment of acidophilic, basophilic, or proline-directed kinase substrates, the method is applicable to identify targets of all these three types of protein kinases. The MKK7-MPK3/6 module, for example, is involved in the regulation of plant development and plant basal and systemic immune responses, but little is known about downstream cascade components. Using our here described phosphoproteomics approach we identified several MPK substrates downstream of the MKK7-MPK3/6 phosphorylation cascade in Arabidopsis. The identification and validation of dynamin-related protein 2 as a novel phosphorylation substrate of the MKK7-MPK3/6 module establishes a novel link between MPK signaling and clathrin-mediated vesicle trafficking