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Abstract Background Protein nanostructures produced through the self-assembly of individual subunits are attractive scaffolds to attach and position functional molecules for applications in biomaterials, metabolic engineering, tissue engineering, and a plethora of nanomaterials. However, the assembly of multicomponent protein nanomaterials is generally a laborious process that requires each protein component to be separately expressed and purified prior to assembly. Moreover, excess components not incorporated into the final assembly must be removed from the solution and thereby necessitate additional processing steps. Results We developed an efficient approach to purify functionalized protein nanostructures directly from bacterial lysates through a type of multimodal chromatography (MMC) that combines size-exclusion, hydrophilic interaction, and ion exchange to separate recombinant protein assemblies from excess free subunits and bacterial proteins. We employed the ultrastable filamentous protein gamma-prefoldin as a material scaffold that can be functionalized with a variety of protein domains through SpyTag/SpyCatcher conjugation chemistry. The purification of recombinant gamma-prefoldin filaments from bacterial lysates using MMC was tested across a wide range of salt concentrations and pH, demonstrating that the MMC resin is robust, however the optimal choice of salt species, salt concentration, and pH is likely dependent on the protein nanostructure to be purified. In addition, we show that pre-processing of the samples with tangential flow filtration to remove nucleotides and metabolites improves resin capacity, and that post-processing with Triton X-114 phase partitioning is useful to remove lipids and any remaining lipid-associated protein. Subsequently, functionalized protein filaments were purified from bacterial lysates using MMC and shown to be free of unincorporated subunits. The assembly and purification of protein filaments with varying amounts of functionalization was confirmed using polyacrylamide gel electrophoresis, Förster resonance energy transfer, and transmission electron microscopy. Finally, we compared our MMC workflow to anion exchange chromatography with the purification of encapsulin nanocompartments containing a fluorescent protein as a cargo, demonstrating the versatility of the protocol and that the purity of the assembly is comparable to more traditional procedures. Conclusions We envision that the use of MMC will increase the throughput of protein nanostructure prototyping as well as enable the upscaling of the bioproduction of protein nanodevices. Graphic Abstract

Abstract Background DNA methylation is a critical molecular mark involved in cellular differentiation and cell-specific processes. Single-cell whole genome DNA methylation profiling methods hold great potential to resolve the DNA methylation profiles of individual cell-types. Here we present a method that couples single-cell combinatorial indexing (sci) with enzymatic conversion (sciEM) of unmethylated cytosines. Results The sciEM method facilitates DNA methylation profiling of single-cells that is highly correlated with single-cell bisulfite-based workflows (r2 > 0.99) whilst improving sequencing alignment rates, reducing adapter contamination and over-estimation of DNA methylation levels (CpG and non-CpG). As proof-of-concept we perform sciEM analysis of the temporal lobe, motor cortex, hippocampus and cerebellum of the human brain to resolve single-cell DNA methylation of all major cell-types. Conclusion To our knowledge sciEM represents the first non-bisulfite single-cell DNA methylation sequencing approach with single-base resolution.

Growth Differentiation Factor-15 (GDF15) is a divergent TGF-beta superfamily cytokine that is overexpressed by most cancers and is induced by anticancer therapy. Transgenic and induced animal models suggest that it protects from cancer development but the mechanisms are uncertain. We investigated the role of immunity in GDF15 induced reduction in prostate cancer (PCa) growth. The C57BL/6 transgenic TRAMP prostate cancer prone mice were bred with mice that were immunodeficient and/or systemically overexpressed GDF15. We developed a novel orthotopic TRAMP PCa model in which primary TRAMP tumor cells were implanted into prostates of mice to reduce the study time. These mice were administered recombinant mouse GDF15, antibody to CD8, PD1 or their respective controls. We found that GDF15 induced protection from tumor growth was reversed by lack of adaptive immunity. Flow cytometric evaluation of lymphocytes within these orthotopic tumors showed that GDF15 overexpression was associated with increased CD8 T cell numbers and an increased number and proportion of recently activated CD8+CD11c+ T cells and a reduced proportion of "exhausted" CD8+PD1+ T cells. Further, depletion of CD8 T cells in tumor bearing mice abolished the GDF15 induced protection from tumor growth. Infusion of GDF15 into mice bearing orthotopic TRAMP tumor, substantially reduced tumor growth that was further reduced by concurrent PD1 antibody administration. GDF15 overexpression or recombinant protein protects from TRAMP tumor growth by modulating CD8 T cell mediated antitumor immunity and augments the positive effects of anti-PD1 blockers.

Drug resistance among parasitic nematodes has resulted in an urgent need for the development of new therapies. However, the high re-discovery rate of anti-nematode compounds from terrestrial environments necessitates a new repository for future drug research. Marine epiphytes are hypothesised to produce nematicidal compounds as a defence against bacterivorous predators, thus representing a promising yet underexplored source for anti-nematode drug discovery. The marine epiphytic bacterium Pseudoalteromonas tunicata is known to produce several bioactive compounds. Screening heterologously expressed genomic libraries of P. tunicata against the nematode Caenorhabditis elegans, identified as an E. coli clone (HG8), shows fast-killing activity. Here we show that clone HG8 produces a novel nematode-killing protein-1 (Nkp-1) harbouring a predicted carbohydrate-binding domain with weak homology to known bacterial pore-forming toxins. We found bacteria expressing Nkp-1 were able to colonise the C. elegans intestine, with exposure to both live bacteria and protein extracts resulting in physical damage and necrosis, leading to nematode death within 24 h of exposure. Furthermore, this study revealed C. elegans dar (deformed anal region) and internal hatching may act as a nematode defence strategy against Nkp-1 toxicity. The characterisation of this novel protein and putative mode of action not only contributes to the development of novel anti-nematode applications in the future but reaffirms the potential of marine epiphytic bacteria as a new source of novel biomolecules.

Summary We report herein the purification of a chloroform (CF)‐reducing enzyme, TmrA, from the membrane fraction of a strict anaerobe Dehalobacter sp. strain UNSWDHB to apparent homogeneity with an approximate 23‐fold increase in relative purity compared to crude lysate. The membrane fraction obtained by ultracentrifugation was solubilized in Triton X‐100 in the presence of glycerol, followed by purification by anion exchange chromatography. The molecular mass of the purified TmrA was determined to be 44.5 kDa by SDS‐PAGE and MALDI‐TOF/TOF. The purified dehalogenase reductively dechlorinated CF to dichloromethane in vitro with reduced methyl viologen as the electron donor at a specific activity of (1.27 ± 0.04) × 103 units mg protein−1. The optimum temperature and pH for the activity were 45°C and 7.2, respectively. The UV‐visible spectrometric analysis indicated the presence of a corrinoid and two [4Fe‐4S] clusters, predicted from the amino acid sequence. This is the first report of the production, purification and biochemical characterization of a CF reductive dehalogenase.