Characterization of Spore Proteins Using a Nanoscaffold Vaccine Platform.

Subunit vaccines are theoretically safe and easy to manufacture but require effective adjuvants and delivery systems to yield protective immunity, particularly at critical mucosal sites such as the lung. We investigated nanolipoprotein particles (NLPs) containing the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) as a platform for intranasal vaccination against . Modified lipids enabled attachment of disparate spore and toxin protein antigens.

Coordinated Assembly of the Bacillus anthracis Coat and Exosporium during Bacterial Spore Outer Layer Formation.

Bacterial spores produced by the are composed of concentric shells, each of which contributes to spore function. Spores from all species possess a cortex and coat, but spores from many species possess additional outer layers. The outermost layer of spores, the exosporium, is separated from the coat by a gap known as the interspace.

Enhancement of antigen-specific CD4 and CD8 T cell responses using a self-assembled biologic nanolipoprotein particle vaccine.

To address the need for vaccine platforms that induce robust cell-mediated immunity, we investigated the potential of utilizing self-assembling biologic nanolipoprotein particles (NLPs) as an antigen and adjuvant delivery system to induce antigen-specific murine T cell responses. We utilized OT-I and OT-II TCR-transgenic mice to investigate the effects of NLP-mediated delivery of the model antigen ovalbumin (OVA) on T cell activation. Delivery of OVA with the TLR4 agonist monophosphoryl lipid A (MPLA) in the context of NLPs significantly enhanced the activation of both CD4 and CD8 T cells in vitro compared to co-administration of free OVA and MPLA.

Novel 6xHis tagged foot-and-mouth disease virus vaccine bound to nanolipoprotein adjuvant via metal ions provides antigenic distinction and effective protective immunity.

Here, we engineered two FMD viruses with histidine residues inserted into or fused to the FMDV capsid. Both 6xHis viruses exhibited growth kinetics, plaque morphologies and antigenic characteristics similar to wild-type virus. The 6xHis tag allowed one-step purification of the mutant virions by Co(2+) affinity columns.

Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles.

Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon.

Colocalized delivery of adjuvant and antigen using nanolipoprotein particles enhances the immune response to recombinant antigens.

Subunit antigen-based vaccines can provide a number of important benefits over traditional vaccine candidates, such as overall safety. However, because of the inherently low immunogenicity of these antigens, methods for colocalized delivery of antigen and immunostimulatory molecules (i.e.

Repurposing screens identify rifamycins as potential broad-spectrum therapy for multidrug-resistant Acinetobacter baumannii and select agent microorganisms.

Aims: Estimates suggest that the drug discovery and development processes take between 10 and 15 years, with costs ranging between US$500 million and $2 billion. A growing number of bacteria have become resistant to approved antimicrobials. For example, the Gram-negative bacterium Acinetobacter baumannii has become multidrug resistant (MDR) and is now an important pathogen to the US military in terms of wound infections.

High-throughput isotopic analysis of RNA microarrays to quantify microbial resource use.

Most microorganisms remain uncultivated, and typically their ecological roles must be inferred from diversity and genomic studies. To directly measure functional roles of uncultivated microbes, we developed Chip-stable isotope probing (SIP), a high-sensitivity, high-throughput SIP method performed on a phylogenetic microarray (chip). This approach consists of microbial community incubations with isotopically labeled substrates, hybridization of the extracted community rRNA to a microarray and measurement of isotope incorporation--and therefore substrate use--by secondary ion mass spectrometer imaging (NanoSIMS).

Isolation, characterization, and stability of discretely-sized nanolipoprotein particles assembled with apolipophorin-III.

Background: Nanolipoprotein particles (NLPs) are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. NLPs assembled with human apolipoproteins have been used for myriad biotechnology applications, including membrane protein solubilization, drug delivery, and diagnostic imaging. To expand the repertoire of lipoproteins for these applications, insect apolipophorin-III (apoLp-III) was evaluated for the ability to form discretely-sized, homogeneous, and stable NLPs.

Kinetic analysis of his-tagged protein binding to nickel-chelating nanolipoprotein particles.

Nanolipoprotein particles (NLPs) are discoidal self-assembling membrane mimetics that have been primarily used as a platform for the solubilization and stabilization of membrane proteins. Nickel-chelating nanolipoprotein particles (NiNLPs) containing nickel-chelating lipids (Ni-lipid) for the targeted immobilization of His-tagged proteins hold promise as carriers of hydrophilic biological molecules for a range of applications. The effect of protein loading (i.

Conjugation to nickel-chelating nanolipoprotein particles increases the potency and efficacy of subunit vaccines to prevent West Nile encephalitis.

Subunit antigens are attractive candidates for vaccine development, as they are safe, cost-effective, and rapidly produced. Nevertheless, subunit antigens often need to be adjuvanted and/or formulated to produce products with acceptable potency and efficacy. Here, we describe a simple method for improving the potency and efficacy of a recombinant subunit antigen by its immobilization on nickel-chelating nanolipoprotein particles (NiNLPs).

Characterization and purification of polydisperse reconstituted lipoproteins and nanolipoprotein particles.

Heterogeneity is a fact that plagues the characterization and application of many self-assembled biological constructs. The importance of obtaining particle homogeneity in biological assemblies is a critical goal, as bulk analysis tools often require identical species for reliable interpretation of the results-indeed, important tools of analysis such as x-ray diffraction typically require over 90% purity for effectiveness. This issue bears particular importance in the case of lipoproteins.

Hydrogen production by a hyperthermophilic membrane-bound hydrogenase in water-soluble nanolipoprotein particles.

Hydrogenases constitute a promising class of enzymes for ex vivo hydrogen production. Implementation of such applications is currently hindered by oxygen sensitivity and, in the case of membrane-bound hydrogenases (MBHs), poor water solubility. Nanolipoprotein particles (NLPs) formed from apolipoproteins and phospholipids offer a novel means of incorporating MBHs into a well-defined water-soluble matrix that maintains the enzymatic activity and is amenable to incorporation into more complex architectures.

Immobilization of His-tagged proteins on nickel-chelating nanolipoprotein particles.

Nanolipoprotein particles (NLPs) are nanometer-sized, discoidal particles that self-assemble from purified apolipoprotein and phospholipid. Their size and facile functionalization suggest potential application of NLPs as platforms for the presentation and delivery of recombinant proteins. To this end, we investigated incorporation of nickel-chelating lipids into NLPs (NiNLPs) and subsequent sequestration of polyhistidine (His)-tagged proteins.

Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles.

To better understand the incorporation of membrane proteins into discoidal nanolipoprotein particles (NLPs) we have used atomic force microscopy (AFM) to image and analyze NLPs assembled in the presence of bacteriorhodopsin (bR), lipoprotein E4 n-terminal 22k fragment scaffold and DMPC lipid. The self-assembly process produced two distinct NLP populations: those containing inserted bR (bR-NLPs) and those that did not (empty-NLPs). The bR-NLPs were distinguishable from empty-NLPs by an average increase in height of 1.

Cell-free expression for nanolipoprotein particles: building a high-throughput membrane protein solubility platform.

Membrane-associated proteins and protein complexes account for approximately a third or more of the proteins in the cell (1, 2). These complexes mediate essential cellular processes; including signal transduc-tion, transport, recognition, bioenergetics and cell-cell communication. In general, membrane proteins are challenging to study because of their insolubility and tendency to aggregate when removed from their protein lipid bilayer environment.

Cell-free co-expression of functional membrane proteins and apolipoprotein, forming soluble nanolipoprotein particles.

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs.

Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach.

We report a cell-free approach for expressing and inserting integral membrane proteins into water-soluble particles composed of discoidal apolipoprotein-lipid bilayers. Proteins are inserted into the particles, circumventing the need of extracting and reconstituting the product into membrane vesicles. Moreover, the planar nature of the membrane support makes the protein freely accessible from both sides of the lipid bilayer.

Quantifying size distributions of nanolipoprotein particles with single-particle analysis and molecular dynamic simulations.

Self-assembly of purified apolipoproteins and phospholipids results in the formation of nanometer-sized lipoprotein complexes, referred to as nanolipoprotein particles (NLPs). These bilayer constructs are fully soluble in aqueous environments and hold great promise as a model system to aid in solubilizing membrane proteins. Size variability in the self-assembly process has been recognized for some time, yet limited studies have been conducted to examine this phenomenon.

Amphotericin B induces interdigitation of apolipoprotein stabilized nanodisk bilayers.

Amphotericin B nanodisks (AMB-ND) are ternary complexes of AMB, phospholipid and apolipoprotein organized as discrete nanometer scale disk-shaped bilayers. In gel filtration chromatography experiments, empty ND lacking AMB elute as a single population of particles with a molecular weight in the range of 200 kDa. AMB-ND formulated at a 4:1 phospholipid:AMB weight ratio separated into two peaks.

Different apolipoproteins impact nanolipoprotein particle formation.

Spontaneous interaction of purified apolipoproteins and phospholipids results in formation of lipoprotein particles with nanometer-sized dimensions; we refer to these assemblies as nanolipoprotein particles or NLPs. These bilayer constructs can serve as suitable mimetics of biological membranes and are fully soluble in aqueous environments. We made NLPs from dimyristoylphospatidylcholine (DMPC) in combination with each of four different apolipoproteins: apoA-I, Delta-apoA-I fragment, apoE4 fragment, and apolipophorin III (apoLp-III) from the silk moth B.

Peptide stabilized amphotericin B nanodisks.

Nanometer scale apolipoprotein A-I stabilized phospholipid disk complexes (nanodisks; ND) have been formulated with the polyene antibiotic amphotericin B (AMB). The present studies were designed to evaluate if a peptide can substitute for the function of the apolipoprotein component of ND with respect to particle formation and stability. An 18-residue synthetic amphipathic alpha-helical peptide, termed 4F (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH(2)), solubilized vesicles comprised of egg phosphatidylcholine (egg PC), dipentadecanoyl PC or dimyristoylphosphatidylcholine (DMPC) at rates greater than or equal to solubilization rates observed with human apolipoprotein A-I (apoA-I; 243 amino acids).