Lebhar, H. | bioRxiv Science

bioRxiv2022

Single-cell DNA methylation sequencing by combinatorial indexing and enzymatic DNA methylation conversion.

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. The method facilitates single-base resolution 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.

genomics↗

bioRxiv2022

Single-step purification of functionalized protein nanostructures using multimodal chromatography

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. Here, we developed an efficient approach to purify functionalized protein filament assemblies directly from bacterial lysates in a single step through a type of multimodal chromatography that combines size-exclusion, hydrophilic interaction, and ion exchange to separate recombinant protein assemblies from excess free subunits and bacterial proteins. In this approach, the ultrastable filamentous protein gamma-prefoldin was employed 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 multimodal chromatography was optimized across a wide range of salt concentrations and pH. Subsequently, functionalized protein assemblies were purified from bacterial lysates using multimodal chromatography in a single step and shown to befree of unincorporated subunits. The assembly and purification of protein nanostructures with varying amounts of functionalization was confirmed using polyacrylamide gel electrophoresis, Forster resonance energy transfer, and transmission electron microscopy. We envision that the use of multimodal chromatography will increase the throughput of protein nanostructure prototyping as well as enable the upscaling of the bioproduction of protein nanodevices.

bioengineering↗