Upscaling and consistent sialylation of plant produced antibodies

Mestrado em Engenharia Agronómica - Instituto Superior de AgronomiaGlycoproteins are a major part of biopharmaceutical products. For their production, a high level of homogeny is required, not only for the protein but also for attached glycans, since these oligossacharides influence protein function. For this goal, plants emerge as an ideal biopharmaceutical production platform. By infiltrating adult plant leaves with a suspension of Agrobacterium tumefaciens containing a plasmid for the gene of interest, a high amount of protein can be produced in a short period. In this study, the production of two antibodies in genetically glycoengineered Nicotiana benthamiana was described. These antibodies, Rituximab and KBPA, served as models for Immunoglobulin groups G and M. Additionally, co-infiltration of the genes for these antibodies with glycoengineering constructs allowed for specific glycan structures attached to these proteins. Finally, the down-regulation of β-N-acetylhexosaminidase 3 with RNAi technology was described, inhibiting the effect of this glycosidase on plant produced proteins

Phylogeography and modes of reproduction in diploid and tetraploid halophytes of Limonium species (Plumbaginaceae): evidence for a pattern of geographical parthenogenesis

Background and Aims The genus Limonium (Plumbaginaceae) has long been recognized to have sexual and apomictic (asexual seed formation) modes of reproduction. This study aimed to elucidate phylogeographical patterns and modes of reproduction in diploid and tetraploid Limonium species, namely three putative sexual diploid species with morphological affinities (L. nydeggeri, L. ovalifolium, L. lanceolatum) and three related, probably apomict tetraploid species (L. binervosum, L. dodartii, L. multiflorum). Methods cpDNA diversity and differentiation between natural populations of the species were investigated using two chloroplast sequence regions (trnL intron and trnL–trnF intergenic spacer). Floral heteromorphies, ovule cytoembryological analyses and pollination and crossing tests were performed in representative species of each ploidy group, namely diploid L. ovalifolium and tetraploid L. multiflorum, using plants from greenhouse collections. Key Results and Conclusions Genetic analyses showed that diploid species have a higher haplotype diversity and a higher number of unique (endemic) haplotypes than tetraploid species. Network analysis revealed correlations between cpDNA haplotype distribution and ploidy groups, species groups and geographical origin, and haplotype sharing within and among species with distinct ploidy levels. Reproductive biology analyses showed that diploid L. ovalifolium mainly forms meiotically reduced tetrasporic embryo sacs of Gagea ova, Adoxa and Drusa types. Limonium multiflorum, however, has only unreduced, diplosporic (apomictic) embryo sacs of Rudbeckia type, and autonomous apomictic development seems to occur. Taken together, the findings provide evidence of a pattern of ‘geographical parthenogenesis’ in which quaternary climatic oscillations appear to be involved in the geographical patterns of coastal diploid and tetraploid Limonium speciesinfo:eu-repo/semantics/publishedVersio

Stabilization of the CD81 Large Extracellular Loop with De Novo Disulfide Bonds Improves Its Amenability for Peptide Grafting

Tetraspan proteins are significantly enriched in the membranes of exosomal vesicles (EVs) and their extracellular domains are attractive targets for engineering towards specific antigen recognition units. To enhance the tolerance of a tetraspanin fold to modification, we achieved significant thermal stabilization of the human CD81 large extracellular loop (hCD81 LEL) via de novo disulfide bonds. The best mutants were shown to exhibit a positive shift in the melting temperature (Tm) of up to 25 °C. The combination of two most potent disulfide bonds connecting different strands of the protein resulted in a mutant with a Tm of 109 °C, 43 °C over the Tm of the wild-type hCD81 LEL. A peptide sequence binding to the human transferrin receptor (hTfr) was engrafted into the D-segment of the hCD81 LEL, resulting in a mutant that still exhibited a compact fold. Grafting of the same peptide sequence between helices A and B resulted in a molecule with an aberrant profile in size exclusion chromatography (SEC), which could be improved by a de novo cysteine bond connecting both helices. Both peptide-grafted proteins showed an enhanced internalization into the cell line SK-BR3, which strongly overexpresses hTfr. In summary, the tetraspan LEL fold could be stabilized to enhance its amenability for engineering into a more versatile protein scaffold