TY - JOUR A1 - Ortiz-Soto, Maria Elena A1 - Seibel, Jürgen T1 - Expression of Functional Human Sialyltransferases ST3Gal1 and ST6Gal1 in Escherichia coli JF - PLoS ONE N2 - Sialyltransferases (STs) are disulfide-containing, type II transmembrane glycoproteins that catalyze the transfer of sialic acid to proteins and lipids and participate in the synthesis of the core structure oligosaccharides of human milk. Sialic acids are found at the outermost position of glycostructures, playing a key role in health and disease. Sialylation is also essential for the production of recombinant therapeutic proteins (RTPs). Despite their importance, availability of sialyltransferases is limited due to the low levels of stable, soluble and active protein produced in bacterial expression systems, which hampers biochemical and structural studies on these enzymes and restricts biotechnological applications. We report the successful expression of active human sialyltransferases ST3Gal1 and ST6Gal1 in commercial Escherichia coli strains designed for production of disulfide-containing proteins. Fusion of hST3Gal1 with different solubility enhancers and substitution of exposed hydrophobic amino acids by negatively charged residues (supercharging-like approach) were performed to promote solubility and folding. Co-expression of sialyltransferases with the chaperon/foldases sulfhydryl oxidase, protein disulfide isomerase and disulfide isomerase C was explored to improve the formation of native disulfide bonds. Active sialyltransferases fused with maltose binding protein (MBP) were obtained in sufficient amounts for biochemical and structural studies when expressed under oxidative conditions and co-expression of folding factors increased the yields of active and properly folded sialyltransferases by 20%. Mutation of exposed hydrophobic amino acids increased recovery of active enzyme by 2.5-fold, yielding about 7 mg of purified protein per liter culture. Functionality of recombinant enzymes was evaluated in the synthesis of sialosides from the β-d-galactoside substrates lactose, N-acetyllactosamine and benzyl 2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl)-α-d-galactopyranoside. KW - cytoplasm KW - recombinant proteins KW - solubility KW - disulfide bonds KW - enzymes KW - enzyme purification KW - enzyme structure KW - sialic acids Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-179807 VL - 11 IS - 5 ER - TY - JOUR A1 - Beiss, Veronique A1 - Spiegel, Holger A1 - Boes, Alexander A1 - Scheuermayer, Matthias A1 - Reimann, Andreas A1 - Schillberg, Stefan A1 - Fischer, Rainer T1 - Plant expression and characterization of the transmission-blocking vaccine candidate PfGAP50 JF - BMC Biotechnology N2 - Background: Despite the limited success after decades of intensive research and development efforts, vaccination still represents the most promising strategy to significantly reduce the disease burden in malaria endemic regions. Besides the ultimate goal of inducing sterile protection in vaccinated individuals, the prevention of transmission by so-called transmission blocking vaccines (TBVs) is being regarded as an important feature of an efficient malaria eradication strategy. Recently, Plasmodium falciparum GAP50 (PfGAP50), a 44.6 kDa transmembrane protein that forms an essential part of the invasion machinery (glideosome) multi-protein complex, has been proposed as novel potential transmission-blocking candidate. Plant-based expression systems combine the advantages of eukaryotic expression with a up-scaling potential and a good product safety profile suitable for vaccine production. In this study we investigated the feasibility to use the transient plant expression to produce PfGAP50 suitable for the induction of parasite specific inhibitory antibodies. Results: We performed the transient expression of recombinant PfGAP50 in Nicotiana benthamiana leaves using endoplasmatic reticulum (ER) and plastid targeting. After IMAC-purification the protein yield and integrity was investigated by SDS-PAGE and Western Blot. Rabbit immune IgG derived by the immunization with the plastidtargeted variant of PfGAP50 was analyzed by immune fluorescence assay (IFA) and zygote inhibition assay (ZIA). PfGAP50 could be produced in both subcellular compartments at different yields IMAC (Immobilized Metal Affinity Chromatography) purification from extract yielded up to 4.1 mu g/g recombinant protein per fresh leaf material for ER-retarded and 16.2 mu g/g recombinant protein per fresh leave material for plasmid targeted PfGAP50, respectively. IgG from rabbit sera generated by immunization with the recombinant protein specifically recognized different parasite stages in immunofluorescence assay. Furthermore up to 55 % inhibition in an in vitro zygote inhibition assay could be achieved using PfGAP50-specific rabbit immune IgG. Conclusions: The results of this study demonstrate that the plant-produced PfGAP50 is functional regarding the presentation of inhibitory epitopes and could be considered as component of a transmission-blocking malaria vaccine formulation. KW - PFS25 KW - plastid targeting KW - plant-made vaccines KW - agroinfiltration KW - gametes KW - sexual stage KW - plasmodium falciparum KW - membrane KW - antibodies KW - immunization KW - RTS,S/AS01 malaria vaccine KW - recombinant proteins KW - cost-effectiveness KW - purification Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-137327 VL - 15 IS - 108 ER - TY - JOUR A1 - Biju, Joseph A1 - Schwarz, Roland A1 - Linke, Burkhard A1 - Blom, Jochen A1 - Becker, Anke A1 - Claus, Heike A1 - Goesmann, Alexander A1 - Frosch, Matthias A1 - Müller, Tobias A1 - Vogel, Ulrich A1 - Schoen, Christoph T1 - Virulence Evolution of the Human Pathogen Neisseria meningitidis by Recombination in the Core and Accessory Genome JF - PLoS One N2 - Background Neisseria meningitidis is a naturally transformable, facultative pathogen colonizing the human nasopharynx. Here, we analyze on a genome-wide level the impact of recombination on gene-complement diversity and virulence evolution in N. meningitidis. We combined comparative genome hybridization using microarrays (mCGH) and multilocus sequence typing (MLST) of 29 meningococcal isolates with computational comparison of a subset of seven meningococcal genome sequences. Principal Findings We found that lateral gene transfer of minimal mobile elements as well as prophages are major forces shaping meningococcal population structure. Extensive gene content comparison revealed novel associations of virulence with genetic elements besides the recently discovered meningococcal disease associated (MDA) island. In particular, we identified an association of virulence with a recently described canonical genomic island termed IHT-E and a differential distribution of genes encoding RTX toxin- and two-partner secretion systems among hyperinvasive and non-hyperinvasive lineages. By computationally screening also the core genome for signs of recombination, we provided evidence that about 40% of the meningococcal core genes are affected by recombination primarily within metabolic genes as well as genes involved in DNA replication and repair. By comparison with the results of previous mCGH studies, our data indicated that genetic structuring as revealed by mCGH is stable over time and highly similar for isolates from different geographic origins. Conclusions Recombination comprising lateral transfer of entire genes as well as homologous intragenic recombination has a profound impact on meningococcal population structure and genome composition. Our data support the hypothesis that meningococcal virulence is polygenic in nature and that differences in metabolism might contribute to virulence. KW - population genetics KW - DNA recombination KW - meningococcal disease KW - recombinant proteins KW - genomic databases KW - comparative genomics KW - neisseria meningitidis KW - homologous recombination Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-137960 VL - 6 IS - 4 ER - TY - JOUR A1 - Dombert, Benjamin A1 - Sivadasan, Rajeeve A1 - Simon, Christian M. A1 - Jablonka, Sibylle A1 - Sendtner, Michael T1 - Presynaptic Localization of Smn and hnRNP R in Axon Terminals of Embryonic and Postnatal Mouse Motoneurons N2 - Spinal muscular atrophy (SMA) is caused by deficiency of the ubiquitously expressed survival motoneuron (SMN) protein. SMN is crucial component of a complex for the assembly of spliceosomal small nuclear ribonucleoprotein (snRNP) particles. Other cellular functions of SMN are less characterized so far. SMA predominantly affects lower motoneurons, but the cellular basis for this relative specificity is still unknown. In contrast to nonneuronal cells where the protein is mainly localized in perinuclear regions and the nucleus, Smn is also present in dendrites, axons and axonal growth cones of isolated motoneurons in vitro. However, this distribution has not been shown in vivo and it is not clear whether Smn and hnRNP R are also present in presynaptic axon terminals of motoneurons in postnatal mice. Smn also associates with components not included in the classical SMN complex like RNA-binding proteins FUS, TDP43, HuD and hnRNP R which are involved in RNA processing, subcellular localization and translation. We show here that Smn and hnRNP R are present in presynaptic compartments at neuromuscular endplates of embryonic and postnatal mice. Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo. We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons. These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis. KW - axons KW - spinal cord KW - cytosol KW - DAPI staining KW - immunoprecipitation KW - recombinant proteins KW - protein interactions KW - thoracic diaphragm Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113655 ER - TY - JOUR A1 - Schneider-Schaulies, Jürgen A1 - Bieringer, Maria A1 - Han, Jung Woo A1 - Kendl, Sabine A1 - Khosravi, Mojtaba A1 - Plattet, Philippe T1 - Experimental Adaptation of Wild-Type Canine Distemper Virus (CDV) to the Human Entry Receptor CD150 JF - PLoS ONE N2 - Canine distemper virus (CDV), a close relative of measles virus (MV), is widespread and well known for its broad host range. When the goal of measles eradication may be achieved, and when measles vaccination will be stopped, CDV might eventually cross the species barrier to humans and emerge as a new human pathogen. In order to get an impression how fast such alterations may occur, we characterized required adaptive mutations to the human entry receptors CD150 (SLAM) and nectin-4 as first step to infect human target cells. Recombinant wild-type CDV-A75/17red adapted quickly to growth in human H358 epithelial cells expressing human nectin-4. Sequencing of the viral attachment proteins (hemagglutinin, H, and fusion protein, F) genes revealed that no adaptive alteration was required to utilize human nectin-4. In contrast, the virus replicated only to low titres (102 pfu/ml) in Vero cells expressing human CD150 (Vero-hSLAM). After three passages using these cells virus was adapted to human CD150 and replicated to high titres (105 pfu/ml). Sequence analyses revealed that only one amino acid exchange in the H-protein at position 540 Asp→Gly (D540G) was required for functional adaptation to human CD150. Structural modelling suggests that the adaptive mutation D540G in H reflects the sequence alteration from canine to human CD150 at position 70 and 71 from Pro to Leu (P70L) and Gly to Glu (G71E), and compensates for the gain of a negative charge in the human CD150 molecule. Using this model system our data indicate that only a minimal alteration, in this case one adaptive mutation, is required for adaptation of CDV to the human entry receptors, and help to understand the molecular basis why this adaptive mutation occurs. KW - antibodies KW - canine distemper virus KW - measles virus KW - microbial mutation KW - protein sequencing KW - recombinant proteins KW - ultraviolet radiation KW - vero cells Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96537 ER -