Re-directing bacterial microcompartment systems to enhance recombinant expression of lysis protein E from bacteriophage ϕX174 in Escherichia coli

BackgroundRecombinant expression of toxic proteins remains a challenging problem. One potential method to shield toxicity and thus improve expression of these proteins is to encapsulate them within protein compartments to sequester them away from their targets. Many bacteria naturally produce so-called bacterial microcompartments (BMCs) in which enzymes comprising a biosynthetic pathway are encapsulated in a proteinaeous shell, which is in part thought to shield the cells from the toxicity of reaction intermediates. As a proof-of-concept, we attempted to encapsulate toxic, lysis protein E (E) from bacteriophage ϕX174 inside recombinant BMCs to enhance its expression and achieve higher yields during downstream purification.ResultsE was fused with various N-terminal BMC targeting tags (PduP-, PduD-, and EutC-tags, 18-20 amino acids) and co-expressed with appropriate BMC shell proteins that associate with the tags and are required to form BMCs. Only BMC targeted E fusions, but not non-tagged E, could be successfully cloned, suggesting that the BMC tags reduce the toxicity of E. A PduP-tagged E system appeared to achieve the highest expression of E. Co-expression of Pdu BMC shell proteins with PduP-E increased its expression by 20-50%. Affinity purification of PduP-E via Ni-NTA in the presence of Empigen BB detergent yielded 270 µg of PduP-E per L of induced culture. Removal of the PduP-tag via proteolysis resulted in a final yield of 200 µg of E per L of induced culture, a nearly order of magnitude (~sevenfold) improvement compared to prior reports.ConclusionsThese results demonstrate improved expression of ϕX174 lysis protein E via re-directed BMC systems and ultimately higher E purification yields. Similar strategies can be used to enhance expression of other toxic proteins in recombinant Escherichia coli systems

Evaluation of Inactivation Methods for Rift Valley Fever Virus in Mouse Microglia

Rift Valley fever phlebovirus (RVFV) is a highly pathogenic mosquito-borne virus with bioweapon potential due to its ability to be spread by aerosol transmission. Neurological symptoms are among the worst outcomes of infection, and understanding of pathogenesis mechanisms within the brain is limited. RVFV is classified as an overlap select agent by the CDC and USDA; therefore, experiments involving fully virulent strains of virus are tightly regulated. Here, we present two methods for inactivation of live virus within samples derived from mouse microglia cells using commercially available kits for the preparation of cells for flow cytometry and RNA extraction. Using the flow cytometry protocol, we demonstrate key differences in the response of primary murine microglia to infection with fully virulent versus attenuated RVFV

MAVS mediates a protective immune response in the brain to Rift Valley fever virus

Rift Valley fever virus (RVFV) is a highly pathogenic mosquito-borne virus capable of causing hepatitis, encephalitis, blindness, hemorrhagic syndrome, and death in humans and livestock. Upon aerosol infection with RVFV, the brain is a major site of viral replication and tissue damage, yet pathogenesis in this organ has been understudied. Here, we investigated the immune response in the brain of RVFV infected mice. In response to infection, microglia initiated robust transcriptional upregulation of antiviral immune genes, as well as increased levels of activation markers and cytokine secretion that is dependent on mitochondrial antiviral-signaling protein (MAVS) and independent of toll-like receptors 3 and 7. In vivo, Mavs-/- mice displayed enhanced susceptibility to RVFV as determined by increased brain viral burden and higher mortality. Single-cell RNA sequence analysis identified defects in type I interferon and interferon responsive gene expression within microglia in Mavs-/- mice, as well as dysregulated lymphocyte infiltration. The results of this study provide a crucial step towards understanding the precise molecular mechanisms by which RVFV infection is controlled in the brain and will help inform the development of vaccines and antiviral therapies that are effective in preventing encephalitis

Large-scale application of free energy perturbation calculations for antibody design

Abstract Alchemical free energy perturbation (FEP) is a rigorous and powerful technique to calculate the free energy difference between distinct chemical systems. Here we report our implementation of automated large-scale FEP calculations, using the Amber software package, to facilitate antibody design and evaluation. In combination with Hamiltonian replica exchange, our FEP simulations aim to predict the effect of mutations on both the binding affinity and the structural stability. Importantly, we incorporate multiple strategies to faithfully estimate the statistical uncertainties in the FEP results. As a case study, we apply our protocols to systematically evaluate variants of the m396 antibody for their conformational stability and their binding affinity to the spike proteins of SARS-CoV-1 and SARS-CoV-2. By properly adjusting relevant parameters, the particle collapse problems in the FEP simulations are avoided. Furthermore, large statistical errors in a small fraction of the FEP calculations are effectively reduced by extending the sampling, such that acceptable statistical uncertainties are achieved for the vast majority of the cases with a modest total computational cost. Finally, our predicted conformational stability for the m396 variants is qualitatively consistent with the experimentally measured melting temperatures. Our work thus demonstrates the applicability of FEP in computational antibody design

Cationic HDL mimetics enhance in vivo delivery of self-replicating mRNA

In vivo delivery of large RNA molecules has significant implications for novel gene therapy, biologics delivery, and vaccine applications. We have developed cationic nanolipoprotein particles (NLPs) to enhance the complexation and delivery of large self-amplifying mRNAs (replicons) in vivo. NLPs are high-density lipoprotein (HDL) mimetics, comprised of a discoidal lipid bilayer stabilized by apolipoproteins that are readily functionalized to provide a versatile delivery platform. Herein, we systematically screened NLP assembly with a wide range of lipidic and apolipoprotein constituents, using biophysical metrics to identify lead candidates for in vivo RNA delivery. NLPs formulated with cationic lipids successfully complexed with RNA replicons encoding luciferase, provided measurable protection from RNase degradation, and promoted replicon in vivo expression. The NLP complexation of the replicon and in vivo transfection efficiency were further enhanced by modulating the type and percentage of cationic lipid, the ratio of cationic NLP to replicon, and by incorporating additive molecules

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. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteins as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. These studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses

Anti-YopD western blot confirms YopD complex formation with NLPs.

<p>Purified expressed YopB and/or YopD co-expressed with Δ49ApoA1 in the presence of lipid. <b>A)</b> Native 4–20% Tris-glycine PAGE gel, Native Tris-glycine buffer, detection by SyproRuby fluorescence total protein stain. <b>B)</b> Anti-YopD western blot of a native 4–20% Tris-glycine PAGE gel, Native Tris-glycine buffer, detection by fluorescence of rhodamine conjugated secondary antibody. The lanes are represented as follows: <b>1)</b> YopB with Δ49ApoA1; <b>2)</b> YopB/D with Δ49ApoA1; <b>3)</b> YopD with Δ49ApoA1; <b>4)</b> Δ49ApoA1 only (empty-NLP); <b>NM</b>) Protein mass standard (kDa) NativeMark (Invitrogen).</p