101 research outputs found
Structure determination of Split-soret Cytochrome from a Desulfovibrio species isolated from a human abdominal abcess
The determined structure of the split-soret cytochrome (SSC) isolated from Desulfovibrio desulfuricans ATCC 27774 (D.d.) revealed a new Heme arrangement, which suggests that this protein constitutes a new cytochrome class.. SSC is a 52.6kDa homodimer containing four hemes at one end of the molecule. In each monomer the two hemes have their edges overlapped within van der Waals contacts. The polypeptide chain of each monomer supplies the sixth ligand to the heme-iron of the other monomer. A similar protein was recently purified from a homologous Desulfovibrio clinical strain isolated from an abdominal wall abscess in human patient2. Crystals of this SSC were grown using vapour diffusion method in the presence of agarose gel. Diffraction data were collected using X-ray synchrotron radiation at the ESRF, beamline, ID 14-1. The structure will be solved by molecular replacement using the structure of the D.d. as a starting model
Novel downstream process and analytical tools developed for Influenza VLP vaccine
Vaccination remains the most effective way to prevent the infection with Influenza viruses. However, their constant antigenic drift implies that current human Influenza vaccines need to be annually updated with high inherent costs. Virus-like particles (VLPs) have become widely used as vaccine candidates because of their versatility, immunogenicity, and safety profile.
In this iBET project we are attempting to produce a candidate for a universal vaccine for which 35 different VLPs (mono, trivalent and pentavalent) were purified. Here we describe three recent advances on Influenza VLPs bioprocessing: two new analytical tools and the development of an integrated all filtration purification process, inserted in the âanything but chromatographyâ concept.
The first method is a label-free tool that uses Biolayer interferometry technology applied on an Octet platform to quantify Influenza VLPs at all stages of DSP. Human and avian sialic acid receptors were used, in order to quantify hemagglutinin (HA) content in several mono- and multivalent Influenza VLP strains. The applied method was able to detect and quantify HA from crude sample up to final VLP product with high throughput, real-time results and improved detection limits, when compared to traditional approaches, crucial for in-line monitoring of DSP.
Using a click-chemistry approach that involves Azidohomoalanine incorporation and functionalization, Influenza VLPs were selectively and fluorescently tagged. Taking into account that this chemical tag does not affect particle size, charge and biological activity we report here a valuable tool to online/at-line product monitoring during DSP optimization of virus related biopharmaceuticals. Moreover, using this tool coupled with FACS we were able to discriminate between VLPs and baculovirus, the major impurity of the system.
The proposed all-filtration process will be described, with special focus on the clarification stage, followed by multiple ultrafiltration and diafiltration steps to achieve the needed concentration and purity specifications. Using this all-filtration platform, we are able to speed up the time process, to improve the scale-up and to reduce costs due to the removal of chromatographic steps
Quorum Sensing Primes the Oxidative Stress Response in the Insect Endosymbiont, Sodalis glossinidius
quorum sensing system relies on the function of two regulatory proteins; SogI (a LuxI homolog) synthesizes a signaling molecule, characterized as N-(3-oxohexanoyl) homoserine lactone (OHHL), and SogR1 (a LuxR homolog) interacts with OHHL to modulate transcription of specific target genes. and SOPE. and SOPE indicates the potential for neofunctionalization to occur during the process of genome degeneration
Unique Structure and Stability of HmuY, a Novel Heme-Binding Protein of Porphyromonas gingivalis
Infection, survival, and proliferation of pathogenic bacteria in humans depend on their capacity to impair host responses and acquire nutrients in a hostile environment. Among such nutrients is heme, a co-factor for oxygen storage, electron transport, photosynthesis, and redox biochemistry, which is indispensable for life. Porphyromonas gingivalis is the major human bacterial pathogen responsible for severe periodontitis. It recruits heme through HmuY, which sequesters heme from host carriers and delivers it to its cognate outer-membrane transporter, the TonB-dependent receptor HmuR. Here we report that heme binding does not significantly affect the secondary structure of HmuY. The crystal structure of heme-bound HmuY reveals a new all-β fold mimicking a right hand. The thumb and fingers pinch heme iron through two apical histidine residues, giving rise to highly symmetric octahedral iron co-ordination. The tetrameric quaternary arrangement of the protein found in the crystal structure is consistent with experiments in solution. It shows that thumbs and fingertips, and, by extension, the bound heme groups, are shielded from competing heme-binding proteins from the host. This may also facilitate heme transport to HmuR for internalization. HmuY, both in its apo- and in its heme-bound forms, is resistant to proteolytic digestion by trypsin and the major secreted proteases of P. gingivalis, gingipains K and R. It is also stable against thermal and chemical denaturation. In conclusion, these studies reveal novel molecular properties of HmuY that are consistent with its role as a putative virulence factor during bacterial infection
The Complete Genome Sequence of âCandidatus Liberibacter solanacearumâ, the Bacterium Associated with Potato Zebra Chip Disease
Zebra Chip (ZC) is an emerging plant disease that causes aboveground decline of
potato shoots and generally results in unusable tubers. This disease has led to
multi-million dollar losses for growers in the central and western United States
over the past decade and impacts the livelihood of potato farmers in Mexico and
New Zealand. ZC is associated with âCandidatus
Liberibacter solanacearumâ, a fastidious alpha-proteobacterium that is
transmitted by a phloem-feeding psyllid vector, Bactericera
cockerelli Sulc. Research on this disease has been hampered by a
lack of robust culture methods and paucity of genome sequence information for
âCa. L. solanacearumâ. Here we present the
sequence of the 1.26 Mbp metagenome of âCa. L.
solanacearumâ, based on DNA isolated from potato psyllids. The coding
inventory of the âCa. L. solanacearumâ genome was
analyzed and compared to related Rhizobiaceae to better
understand âCa. L. solanacearumâ physiology and
identify potential targets to develop improved treatment strategies. This
analysis revealed a number of unique transporters and pathways, all potentially
contributing to ZC pathogenesis. Some of these factors may have been acquired
through horizontal gene transfer. Taxonomically, âCa. L.
solanacearumâ is related to âCa. L.
asiaticusâ, a suspected causative agent of citrus huanglongbing, yet many
genome rearrangements and several gene gains/losses are evident when comparing
these two Liberibacter. species. Relative to âCa. L.
asiaticusâ, âCa. L. solanacearumâ probably
has reduced capacity for nucleic acid modification, increased amino acid and
vitamin biosynthesis functionalities, and gained a high-affinity iron transport
system characteristic of several pathogenic microbes
Prevalence and characteristics of sleep apnoea in patients with stable heart failure: Results from a heart failure clinic
Investigating the physiological response of Pichia (Komagataella) pastoris GS115 to the heterologous expression of misfolded proteins using chemostat cultures
Syntheses and Structures of Bridge and Chelate Isomers of Tetraplatinum(II) Cluster Complex with Diethyldithiophosphate Ion (Et 2
Purification of influenza virus-like particles using sulfated cellulose membrane adsorbers
Quaternary structure of flavorubredoxin as revealed by synchrotron radiation small-angle X-ray scattering
Flavodiiron proteins (FDP) are modular enzymes which function as NO and/or O(2) reductases. Although the majority is composed of two structural domains, the homolog found in Escherichia coli, flavorubredoxin, possesses an extra C-terminal module consisting of a linker and a rubredoxin (Rd) domain necessary for interprotein redox processes. In order to investigate the location of the Rd domain with respect to the flavodiiron structural core, small-angle X-ray scattering was used to construct low-resolution structural models of flavorubredoxin. Scattering patterns from the Rd domain, the FDP core, and full-length flavorubredoxin were collected. The latter two species were found to be tetrameric in solution. Ab initio shape reconstruction and rigid-body modeling indicate a peripheral location for the Rd domains, which appear to have weak contacts with the FDP core. This finding suggests that Rd behaves as an independent domain and is freely available to participate in redox reactions with protein partners
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