Production of high-value recombinant proteins in transgenic seeds is an attractive and economically feasible alternative to conventional systems based on mammalian cells and bacteria. In contrast to leaves, seeds allow high-level accumulation of recombinant proteins in a relatively small volume and a stable environment. We demonstrate that single-chain variable fragment (scFv)-Fc antibodies, with N-terminal signal sequence and C-terminal KDEL tag, can accumulate to very high levels as bivalent IgG-like antibodies in Arabidopsis thaliana seeds and illustrate that a plant-produced anti-hepatitis A virus scFv-Fc has similar antigen-binding and in vitro neutralizing activities as the corresponding full-length IgG. As expected, most scFv-Fc produced in seeds contained only oligomannose-type N-glycans, but, unexpectedly, 35-40% was never glycosylated. A portion of the scFv-Fc was found in endoplasmic reticulum (ER)-derived compartments delimited by ribosomeassociated membranes. Additionally, consistent with the glycosylation data, large amounts of the recombinant protein were deposited in the periplasmic space, implying a direct transport from the ER to the periplasmic space between the plasma membrane and the cell wall. Aberrant localization of the ER chaperones calreticulin and binding protein (BiP) and the endogenous seed storage protein cruciferin in the periplasmic space suggests that overproduction of recombinant scFv-Fc disturbs normal ER retention and proteinsorting mechanisms in the secretory pathway.glycosylation ͉ molecular farming ͉ recombinant antibody ͉ subcellular localization T ransgenic plants for the production of high-value recombinant proteins are a promising alternative to conventional recombinant protein production systems, such as bacteria, yeast, animal, and insect cell cultures (1). One of the most important factors driving research in this field is yield improvement, because of its significant impact on economic feasibility (2). Strategies to increase recombinant protein yield in plants include development of better expression cassettes, improvement of protein stability and accumulation by using specific subcellular targeting signals, and development of downstream processing technologies (3). In this perspective, seed-based platforms are particularly attractive because they allow recombinant proteins to stably accumulate at a relatively high concentration in a compact biomass, which is beneficial for extraction and downstream processing (4). By using a seed-specific expression cassette based on the regulatory signals of seed storage proteins of common bean (Phaseolus vulgaris), and by targeting the recombinant protein to the endoplasmic reticulum (ER), we obtained the highest yields of recombinant proteins in plants described so far: a single-chain variable fragment (scFv) accumulated to levels in excess of 36% of total soluble protein (TSP) in homozygous Arabidopsis seeds, while retaining its antigen-binding activity and affinity (5).For some applications, fusion of the scFv with the Fc doma...

In the present paper, 26 food waste streams were selected according to their exploitation potential and investigated in terms of pectin content. The isolated pectin, subdivided into calcium bound and alkaline extractable pectin, was fully characterized in terms of uronic acid and other sugar composition, methylation and acetylation degree. It was shown that many waste streams can be a valuable source of pectin, but also that pectin structures present a huge structural diversity, resulting in a broad range of pectin structures. These can have different physicochemical and biological properties, which are useful in a wide range of applications. Even if the data could not cover all the possible batch by batch and country variabilities, to date this represents the most complete pectin characterization from food waste streams ever reported in the literature with a homogeneous methodology.

The AFLP technique was used to assess the genetic relationships among the cultivated papaya ( Carica papaya L.) and related species native to Ecuador. Genetic distances based on AFLP data were estimated for 95 accessions belonging to three genera including C. papaya, at least eight Vasconcella species and two Jacaratia species. Cluster analysis using different methods and principal co-ordinate analysis (PCO), based on the AFLP data from 496 polymorphic bands generated with five primer combinations, was performed. The resulted grouping of accessions of each species corresponds largely with their taxonomic classifications and were found to be consistent with other studies based on RAPD, isozyme and cpDNA data. The AFLP analysis supports the recent rehabilitation of the Vasconcella group as a genus; until recently Vasconcella was considered as a section within the genus Carica. Both cluster and PCO analysis clearly separated the species of the three genera and illustrated the large genetic distance between C. papaya accessions and the Vasconcella group. The specific clustering of the highly diverse group of Vasconcella x heilbornii accessions also suggests that these genotypes may be the result of bi-directional introgression events between Vasconcella stipulata and Vasconcella cundinamarcensis.

The chloroplast and mitochondrial DNA diversity of 61 genotypes belonging to 18 Vasconcellea species, the so-called highland papayas, was studied by PCR-RFLP analysis of two non-coding cpDNA regions ( trnM- rbcL and trnK1- trnK2) and one non-coding mtDNA region ( nad4/1- nad4/2). This sample set was supplemented with six genotypes belonging to three other Caricaceae genera: the monotypic genus Carica, including only the cultivated papaya, and the genera Jacaratia and Cylicomorpha. Moringa ovalifolia was added as an outgroup species. The PCR-amplified cpDNA regions were digested with 18 restriction endonucleases, the mtDNA region with 11. A total of 22 point mutations and four insertion/deletions were scored in the sample. A higher level of interspecific variation was detected in the two cpDNA regions in comparison to the analysis of the mtDNA. Wagner parsimony and Neighbor-Joining analysis resulted in dendrograms with similar topologies. PCR-RFLP analysis supported the monophyly of Caricaceae, but among the 26 mutations scored, an insufficient number of markers discriminated between the different Caricaceae genera included in this study. Hence the inference of the intergeneric relationships within Caricaceae was impossible. However, some conclusions can be noted at a lower taxonomic level. The Caricaceae species were divided into two lineages. One group included only Vasconcellea spp., whereas the second included the remaining Vasconcellea spp., together with the papaya genotypes and those from the other Caricaceae genera. This may indicate a higher level of inter-fertility for the Vasconcellea species from the latter clade in interspecific crossings with papaya. The putative progenitors of the natural sterile hybrid V. x heilbornii, i.e. V. stipulata and V. cundinamarcensis, were only distantly related to V. x heilbornii. This indicates that probably none of these species was involved as the maternal progenitor in the origin of V. x heilbornii. Surprisingly, V. x heilbornii had organellar genome patterns identical with V. weberbaueri, suggesting a possible involvement of this species in the origin of V. x heilbornii. On the basis of discrepancy between morphological traits and the cpDNA profiles of some pairs of Vasconcellea species, we believe that besides V. x heilbornii, some other species have originated through interspecific hybridization. A reticulate evolution for Vasconcellea has therefore been suggested. Finally, intraspecific cpDNA variation was detected in V. microcarpa, thus providing molecular evidence for the high diversity previously indicated by morphological observations.

The impact of low-oxygen spiral-filter press technology combined with thermal pasteurization (TP), pulsed electric field (PEF) and high pressure processing (HPP) on cloudy apple juice quality was investigated immediately after the treatments and after 3 weeks of storage at 4 C. Based on equivalent levels of microbial safety and desired shelf-life, low and high processing intensities were selected: TP (72 C/15 s; 85 C/30 s), PEF (12.5 kV/cm, 76.4 kJ/L; 12.3 kV/cm, 132.5 kJ/L), and HPP (400 MPa/3min; 600 MPa/3 min). High intensity thermal treatment resulted in a bright, yellowish color which was maintained during storage. PPO and POD activities were largely reduced by high intensity PEF and TP yet showed high resistance to HPP. The highest vitamin C content was provided by fresh juice followed by PEF-treated juices. Due to oxidative degradation reactions, vitamin C of all treated samples significantly decreased during storage. Immediately after processing, high cloud stability values were obtained in all samples; however, cloud stability decreased during storage particularly for HPP juices with high residual PME. No significant changes were observed in pH, titratable acidity, organic acid and sugar content which also corresponded to sweet and sour taste. Results from untargeted volatile profiles showed that esters increased after PEF and were better retained after HPP. Contrary to TP treatment where ester degradation reactions occurred together with the formation of off-flavors. Most of the volatiles decreased during storage which could be linked to oxidation and ester hydrolysis reactions. Industrial relevanceBeing one of the most popular fruit juices consumed worldwide, cloudy apple juice can still undergo quality changes such as color degradation, cloud loss (fast sedimentation) and flavor changes during processing and storage. This study evaluates the potential of low-oxygen spiral-filter press in combination with different preservation technologies to obtain a maximal quality of cloudy apple juice. Results shows that high intensity thermal pasteurization can effectively inactivate quality-degrading enzymes, therefore it is useful to obtain an optimal cloudy apple juice product in terms of color and cloud stability. Although HPP has minimal impact on aroma of the juice, shelf-life of the juice may be limited due to incomplete enzyme inactivation. In the case of PEF treatment, thermal effects may contribute to maintain apple juice quality.

a b s t r a c tExtracts of the white shaft and green leaves of 30 leek cultivars were investigated for their antioxidant properties, total phenolic (TP) and L-ascorbic acid (AA) content. The measured antioxidant properties included free radical scavenging activities against peroxyl (ORAC) and 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH) and their Fe 3+ reducing capacity (FRAP). The results from this study suggest that the green leek leaves generally have significantly stronger antioxidant properties than the white shaft. Correlation analysis between the TP and the AA content and the antioxidant activity showed that phenolics and ascorbic acid contribute significantly to the antioxidant activity of leek. The three antioxidant activity assays were all correlated for the extracts of the white shaft of the 30 leek cultivars. Principal component analysis (PCA) elucidated the influence of part and type of cultivar on the antioxidant capacity, TP, and Lascorbic acid content, whilst the breeding strategy and seed company had no influence.

SummarySeed-specific expression is an appealing alternative technology for the production of recombinant proteins in transgenic plants. Whereas attractive yields of recombinant proteins have been achieved by this method, little attention has been paid to the intracellular deposition and the quality of such products. Here, we demonstrate a comparative study of two antiviral monoclonal antibodies (mAbs) (HA78 against Hepatitis A virus; 2G12 against HIV) expressed in seeds of Arabidopsis wild-type (wt) plants and glycosylation mutants lacking plant specific N-glycan residues. We demonstrate that 2G12 is produced with complex N-glycans at great uniformity in the wt as well as in the glycosylation mutant, carrying a single dominant glycosylation species, GnGnXF and GnGn, respectively. HA78 in contrast, contains additionally to complex N-glycans significant amounts of oligo-mannosidic structures, which are typical for endoplasmic reticulum (ER)-retained proteins. A detailed subcellular localization study demonstrated the deposition of both antibodies virtually exclusively in the extracellular space, illustrating their efficient secretion. In addition, although a KDELtagged version of 2G12 exhibited an ER-typical N-glycosylation pattern, it was surprisingly detected in protein storage vacuoles. The different antibody variants showed different levels of degradation with hardly any degradation products detectable for HA78 carrying GnGnXF glycans. Finally, we demonstrate functional integrity of the HA78 and 2G12 glycoforms using viral inhibition assays. Our data therefore demonstrate the usability of transgenic seeds for the generation of mAbs with a controlled N-glycosylation pattern, thus expanding the possibilities for the production of optimally glycosylated proteins with enhanced biological activities for the use as human therapeutics.

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