Expression and purification of soluble recombinant full length HIV-1 Pr55(Gag) protein in Escherichia coli

The HIV-1 Gag precursor protein, Pr55(Gag), is a multi-domain polyprotein that drives HIV-1 assembly. The morphological features of HIV-1 suggested Pr55(Gag) assumes a variety of different conformations during virion assembly and maturation, yet structural determination of HIV-1 Pr55(Gag) has not been possible due to an inability to express and to isolate large amounts of full-length recombinant Pr55(Gag) for biophysical and biochemical analyses. This challenge is further complicated by HIV-1 Gag's natural propensity to multimerize for the formation of viral particle (with ∼2500 Gag molecules per virion), and this has led Pr55(Gag) to aggregate and be expressed as inclusion bodies in a number of in vitro protein expression systems. This study reported the production of a recombinant form of HIV-1 Pr55(Gag) using a bacterial heterologous expression system. Recombinant HIV-1 Pr55(Gag) was expressed with a C-terminal His×6 tag, and purified using a combination of immobilized metal affinity chromatography and size exclusion chromatography. This procedure resulted in the production of milligram quantities of high purity HIV-1 Pr55(Gag) that has a mobility that resembles a trimer in solution using size exclusion chromatography analysis. The high quantity and purity of the full length HIV Gag will be suitable for structural and functional studies to further understand the process of viral assembly, maturation and the development of inhibitors to interfere with the process.

One step multifunctional micropatterning of surfaces using asymmetric glow discharge plasma polymerization

Micropatterning of surfaces with varying chemical, physical and topographical properties usually requires a number of fabrication steps. Herein, we describe a micropatterning technique based on plasma enhanced chemical vapour deposition (PECVD) that deposits both protein resistant and protein repellent surface chemistries in a single step. The resulting multifunctional, selective surface chemistries are capable of spatially controlled protein adhesion, geometric confinement of cells and the site specific confinement of enzyme mediated peptide self-assembly.

Protein electrochemistry using graphene-based nano-assembly: an ultrasensitive electrochemical detection of protein molecules via nanoparticle-electrode collisions

We describe a new electrochemical detection approach towards single protein molecules (microperoxidase-11, MP-11), which are attached to the surface of graphene nanosheets. The non-covalently functionalized graphene nanosheets exhibit enhanced electroactive surface area, where amplified redox current is produced when graphene nanosheets collide with the electrode.

Protein electrochemistry using aligned carbon nanotube arrays

The remarkable electrocatalytic properties and small size of carbon nanotubes make them ideal for achieving direct electron transfer to proteins, important in understanding their redox properties and in the development of biosensors. Here, we report shortened SWNTs can be aligned normal to an electrode by self-assembly and act as molecular wires to allow electrical communication between the underlying electrode and redox proteins covalently attached to the ends of the SWNTs, in this case, microperoxidase MP-11. The efficiency of the electron transfer through the SWNTs is demonstrated by electrodes modified with tubes cut to different lengths having the same electron-transfer rate constant.

Lipid rafts and HIV-1 : from viral entry to assembly of progeny virions

Background: Lipid rafts are currently an intensely investigated topic of cell biology. In addition to a demonstrated role in signal transduction of the host cell, lipid rafts serve as entry and exit sites for microbial pathogens and toxins, such as FimH-expressing enterobacteria, influenza virus, measles virus and cholera toxin. Furthermore, caveolae, a specialised form of lipid raft, are required for the conversion of the non-pathogenic prion protein to the pathogenic scrapie isoform.

Objectives: A number of reports have shown, directly or indirectly, that lipid rafts are important at various stages of the human immunodeficiency virus type-1 (HIV-1) replication cycle. The purpose of this paper is to provide a brief overview of the role of membrane-associated lipid rafts in cell biology, and to evaluate how HIV-1 has hijacked this cellular component to support HIV-1 replication. Special sections are devoted to discussing the role of lipid rafts in (1) the entry of HIV-1, (2) signal transduction regulation in HIV-1-infected cells, (3) the trafficking of HIV-1 proteins via lipid rafts during HIV-1 assembly; and a further section discusses the role of cholesterol in mature HIV-1.

Summary: Like a number of other pathogens, HIV-1 has evolved to rely on the host cell lipid rafts to support its propagation during multiple stages of the HIV-1 replication cycle. This review has highlighted the importance of lipid rafts in HIV-1 replication.

Enzyme-assisted self-assembly under thermodynamic control

The production of functional molecular architectures through self-assembly is commonplace in biology, but despite advances^{1, 2, 3}, it is still a major challenge to achieve similar complexity in the laboratory. Self-assembled structures that are reproducible and virtually defect free are of interest for applications in three-dimensional cell culture^{4, 5}, templating⁶, biosensing⁷ and supramolecular electronics⁸. Here, we report the use of reversible enzyme-catalysed reactions to drive self-assembly. In this approach, the self-assembly of aromatic short peptide derivatives^{9, 10} provides a driving force that enables a protease enzyme to produce building blocks in a reversible and spatially confined manner. We demonstrate that this system combines three features: (i) self-correction—fully reversible self-assembly under thermodynamic control; (ii) component-selection—the ability to amplify the most stable molecular self-assembly structures in dynamic combinatorial libraries^{11, 12, 13}; and (iii) spatiotemporal confinement of nucleation and structure growth. Enzyme-assisted self-assembly therefore provides control in bottom-up fabrication of nanomaterials that could ultimately lead to functional nanostructures with enhanced complexities and fewer defects.

Cotranslational assembly of the yeast SET1C histone methyltransferase complex

 While probing the role of RNA for the function of SET1C/COMPASS histone methyltransferase, we identified SET1RC (SET1 mRNA-associated complex), a complex that contains SET1 mRNA and Set1, Swd1, Spp1 and Shg1, four of the eight polypeptides that constitute SET1C. Characterization of SET1RC showed that SET1 mRNA binding did not require associated Swd1, Spp1 and Shg1 proteins or RNA recognition motifs present in Set1. RNA binding was not observed when Set1 protein and SET1 mRNA were derived from independent genes or when SET1 transcripts were restricted to the nucleus. Importantly, the protein-RNA interaction was sensitive to EDTA, to the translation elongation inhibitor puromycin and to the inhibition of translation initiation in prt1-1 mutants. Taken together, our results support the idea that SET1 mRNA binding was dependent on translation and that SET1RC assembled on nascent Set1 in a cotranslational manner. Moreover, we show that cellular accumulation of Set1 is limited by the availability of certain SET1C components, such as Swd1 and Swd3, and suggest that cotranslational protein interactions may exert an effect in the protection of nascent Set1 from degradation.

Non-referenced genome assembly from epigenomic short-read data

Current computational methods used to analyze changes in DNA methylation and chromatin modification rely on sequenced genomes. Here we describe a pipeline for the detection of these changes from short-read sequence data that does not require a reference genome. Open source software packages were used for sequence assembly, alignment, and measurement of differential enrichment. The method was evaluated by comparing results with reference-based results showing a strong correlation between chromatin modification and gene expression. We then used our de novo sequence assembly to build the DNA methylation profile for the non-referenced Psammomys obesus genome. The pipeline described uses open source software for fast annotation and visualization of unreferenced genomic regions from short-read data.

Amyloid peptide self assembly in protic ionic liquids

Overall, this thesis presents the novel approach of using carefully selected PILs to both control amyloid fibril formation and inhibit/dissolve Aβ fibrils through induced helical transitions. These results provide insights into future PIL-mediated protein studies and the potential application of PILs in AD pathogenesis.

De novo assembly and characterization of the invasive Northern Pacific Seastar transcriptome

Invasive species are a major threat to global biodiversity but can also serve as valuable model systems to examine important evolutionary processes. While the ecological aspects of invasions have been well documented, the genetic basis of adaptive change during the invasion process has been hampered by a lack of genomic resources for the majority of invasive species. Here we report the first larval transcriptomic resource for the Northern Pacific Seastar, Asterias amurensis, an invasive marine predator in Australia. Approximately 117.5 million 100 base-pair (bp) paired-end reads were sequenced from a single RNA-Seq library from a pooled set of full-sibling A. amurensis bipinnaria larvae. We evaluated the efficacy of a pre-assembly error correction pipeline on subsequent de novo assembly. Error correction resulted in small but important improvements to the final assembly in terms of mapping statistics and core eukaryotic genes representation. The error-corrected de novo assembly resulted in 115,654 contigs after redundancy clustering. 41,667 assembled contigs were homologous to sequences from NCBI's non-redundant protein and UniProt databases. We assigned Gene Ontology, KEGG Orthology, Pfam protein domain terms and predicted protein-coding sequences to > 36,000 contigs. The final transcriptome dataset generated here provides functional information for 18,319 unique proteins, comprising at least 11,355 expressed genes. Furthermore, we identified 9,739 orthologs to P. miniata proteins, evaluated our annotation pipeline and generated a list of 150 candidate genes for responses to several environmental stressors that may be important for adaptation of A. amurensis in the invasive range. Our study has produced a large set of A. amurensis RNA contigs with functional annotations that can serve as a resource for future comparisons to other echinoderm transcriptomes and gene expression studies. Our data can be used to study the genetic basis of adaptive change and other important evolutionary processes during a successful invasion.

Synthesis and self-assembly behaviour of poly(Nα-Boc-l-tryptophan)-block-poly(ethylene glycol)-block-poly(Nα-Boc-l-tryptophan)

We report for the first time the use of Nα-Boc-l-tryptophan for the synthesis of amphiphilic BAB triblock copolymers for potential drug delivery applications. A library of poly(Nα-Boc-l-tryptophan)-block-poly(ethylene glycol)-block-poly(Nα-Boc-l-tryptophan) (PBoclTrp-b-PEG-b-PBoclTrp) amphiphilic copolymers were synthesized through the ring opening polymerization of Nα-Boc-l-tryptophan Nα-carboxy anhydride as initiated by diamino-terminated PEG of fixed molecular weight (Mn 3350). The influence of the hydrophobic block length over self-assembly was investigated for 4 of the BAB copolymers of molecular weights varying between Mn 5000 and Mn 17000. It was found that an increase in hydrophobic block length led to an increase in hydrodynamic size of aggregates in solution, as well as a decrease in critical micelle concentration. TEM analysis showed the formation of spherical micelles with the largest of the copolymers forming interconnected networks of spherical micelles. The influence of hydrophobic block length over the formation of secondary structure was analyzed using circular dichroism and infrared spectroscopy. Collectively we found that the presence of t-Boc protected l-tryptophan leads to the preferential formation of α-helix secondary structure through hydrogen bonding, which, in a drug delivery vehicle context, could help in controlling drug release. Also, it is believed that the use of novel Nα-Boc-l-tryptophan could improve drug stabilization in the hydrophobic core via π-π interactions between indole rings.