De novo reconstitution of chromatin using wheat germ cell-free protein synthesis.
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ABSTRACT:
SUBMITTER: Endo Y
PROVIDER: S-EPMC8167859 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:One of the biggest bottlenecks for structural analysis of proteins remains the creation of high-yield and high-purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes, while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multiscale eukaryotic wheat germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also report on a robust purification procedure, which can produce highly pure (> 98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis with electron microscopy, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid production and purification of samples that may not be amenable to other recombinant approaches for structural characterization.
| S-EPMC6244559 | biostudies-literature
Project description:Background: Elaboration of the epigenetic regulation of chromatin is a long-standing aim in molecular and cellular biology. Hence, there is a great demand for the development of in vitro methods to reconstitute chromatin that can be used directly for biochemical assays. The widely used wheat germ cell-free protein expression method provides broad applications to investigate the function and structure of eukaryotic proteins. Such advantages, including high translation efficiency, flexibility, and possible automatization, are beneficial for achieving native-like chromatin substrates for in vitro studies.Results: We describe a novel, single-step in vitro chromatin assembly method by using the wheat germ cell-free protein synthesis. We demonstrated that both Drosophila and human chromatins can be reconstituted in the course of the in vitro translation of core histones by the addition of chromatin assembly factors, circular plasmid, and topoisomerase I in an ATP-dependent manner. Drosophila chromatin assembly was performed in 4?h at 26?°C, in the presence of premixed mRNAs encoding the core histones, dAcf1/dISWI chromatin remodeling complex, and nucleosome assembly protein, dNAP1. Similarly, the human chromatin was assembled by co-expressing the human core histones with Drosophila chromatin remodeling factor, dISWI, and chromatin chaperone, dNLP, for 6?h at 26?°C. The presence of reconstituted chromatin was monitored by DNA supercoiling assay, also the regular spacing of nucleosomes was assessed by Micrococcal nuclease assay. Furthermore, Drosophila linker histone H1-containing chromatin was reconstituted, affirming that the in vitro assembled chromatin is suitable for downstream applications.Conclusions: The method described in this study allows the assembly of Drosophila and human chromatins, possibly in native-like form, by using a wheat germ cell-free protein expression. Although both chromatins were reconstituted successfully, there were unexpected differences with respect to the required ratio of histone-coding mRNAs and the reaction time. Overall, our new in vitro chromatin reconstitution method will aid to characterize the unrevealed structure, function, and regulation of chromatin dynamics.
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Project description:Cell-free protein synthesis (CFPS) systems are gaining more importance as universal tools for basic research, applied sciences, and product development with new technologies emerging for their application. Huge progress was made in the field of synthetic biology using CFPS to develop new proteins for technical applications and therapy. Out of the available CFPS systems, wheat germ cell-free protein synthesis (WG-CFPS) merges the highest yields with the use of a eukaryotic ribosome, making it an excellent approach for the synthesis of complex eukaryotic proteins including, for example, protein complexes and membrane proteins. Separating the translation reaction from other cellular processes, CFPS offers a flexible means to adapt translation reactions to protein needs. There is a large demand for such potent, easy-to-use, rapid protein expression systems, which are optimally serving protein requirements to drive biochemical and structural biology research. We summarize here a general workflow for a wheat germ system providing examples from the literature, as well as applications used for our own studies in structural biology. With this review, we want to highlight the tremendous potential of the rapidly evolving and highly versatile CFPS systems, making them more widely used as common tools to recombinantly prepare particularly challenging recombinant eukaryotic proteins.
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Project description:Wheat-germ cell-free protein synthesis (WG-CFPS) is a potent platform for the high-yield production of proteins. It is especially of interest for difficult-to-express eukaryotic proteins, such as toxic and transmembrane proteins, and presents an important tool in high-throughput protein screening. Until recently, an assumed drawback of WG-CFPS was a reduced capacity for post-translational modifications. Meanwhile, phosphorylation has been observed in WG-CFPS; yet, authenticity of the respective phosphorylation sites remained unclear. Here we show that a viral membrane protein, the duck hepatitis B virus (DHBV) large envelope protein (DHBs L), produced by WG-CFPS, is phosphorylated upon translation at the same sites as DHBs L produced during DHBV infection of primary hepatocytes. Furthermore, we show that alternative translation initiation of the L protein, previously identified in virus-producing hepatic cells, occurs on WG-CFPS as well. Together, these findings further strengthen the high potential of WG-CFPS to include the reproduction of specific modifications proteins experience in vivo.
| S-EPMC6902406 | biostudies-literature
Project description:The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification. The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning must be integrated into DNA synthesis methods. Here we show how a new single molecule PCR (smPCR)-based procedure can be employed as a general substitute to in vivo cloning thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our earlier recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligos completely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well.
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Project description:For the first time, we produced four lactoferricin (LFcin) peptides by a cell-free (in vitro) method. These short antimicrobial peptides were expressed in an E. coli cell-free protein synthesis (CFPS) system and the bioactivity of the produced peptides was demonstrated. Additionally, we designed a novel synthetic consensus peptide (ConLFcin). The genes of bovine Lfcin (bLFcin), human Lfcin (hLFcin), camel Lfcin (cLFcin), and ConLFcin were cloned into pET101/D-TOPO vector then peptides were synthesized in vitro by E. coli CFPS system. The antibacterial activity of these synthesized peptides was evaluated against Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA). The four cell-free synthesized peptides showed significant antibacterial potency at minimum inhibitory concentration (MIC) values between 1.25 and 10 μg/mL. cLFcin and ConLFcin showed higher antibacterial effects than bLFcin and hLFcin. Thus, cell-free expression system is an ideal system for rapid expression of functionally active short bioactive peptides.
| S-EPMC7779732 | biostudies-literature
Project description:The availability of inexpensive, on demand synthetic DNA has enabled numerous powerful applications in biotechnology, in turn driving considerable present interest in the de novo synthesis of increasingly longer DNA constructs. The synthesis of DNA from oligonucleotides into products even as large as small viral genomes has been accomplished. Despite such achievements, the costs and time required to generate such long constructs has, to date, precluded gene-length (and longer) DNA synthesis from being an everyday research tool in the same manner as PCR and DNA sequencing. A critical barrier to low-cost, high-throughput de novo DNA synthesis is the frequency at which errors pervade the final product. Here, we employ a DNA mismatch-binding protein, MutS (from Thermus aquaticus) to remove failure products from synthetic genes. This method reduced errors by >15-fold relative to conventional gene synthesis techniques, yielding DNA with one error per 10 000 base pairs. The approach is general, scalable and can be iterated multiple times for greater fidelity. Reductions in both costs and time required are demonstrated for the synthesis of a 2.5 kb gene.
| S-EPMC534640 | biostudies-literature
Project description:BackgroundRapid advances in next-generation sequencing methods have provided new opportunities for transcriptome sequencing (RNA-Seq). The unprecedented sequencing depth provided by RNA-Seq makes it a powerful and cost-efficient method for transcriptome study, and it has been widely used in model organisms and non-model organisms to identify and quantify RNA. For non-model organisms lacking well-defined genomes, de novo assembly is typically required for downstream RNA-Seq analyses, including SNP discovery and identification of genes differentially expressed by phenotypes. Although RNA-Seq has been successfully used to sequence many non-model organisms, the results of de novo assembly from short reads can still be improved by using recent bioinformatic developments.ResultsIn this study, we used 212.6 million pair-end reads, which accounted for 16.2 Gb, to assemble the hexaploid wheat transcriptome. Two state-of-the-art assemblers, Trinity and Trans-ABySS, which use the single and multiple k-mer methods, respectively, were used, and the whole de novo assembly process was divided into the following four steps: pre-assembly, merging different samples, removal of redundancy and scaffolding. We documented every detail of these steps and how these steps influenced assembly performance to gain insight into transcriptome assembly from short reads. After optimization, the assembled transcripts were comparable to Sanger-derived ESTs in terms of both continuity and accuracy. We also provided considerable new wheat transcript data to the community.ConclusionsIt is feasible to assemble the hexaploid wheat transcriptome from short reads. Special attention should be paid to dealing with multiple samples to balance the spectrum of expression levels and redundancy. To obtain an accurate overview of RNA profiling, removal of redundancy may be crucial in de novo assembly.
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Project description:The olefin cross-metathesis reaction allows rapid access to 1,5-dicarbonyl intermediates which, upon treatment with a primary or secondary amine, allow the synthesis of a range of multisubstituted carbocyclic aryl amines. This de novo arene synthesis yields nonclassical substitution patterns in a regioselective and predictable approach that is compatible with several functional groups.
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Project description:The prospect of phasing diffraction data sets ;de novo' for proteins with previously unseen folds is appealing but largely untested. In a first systematic exploration of phasing with Rosetta de novo models, it is shown that all-atom refinement of coarse-grained models significantly improves both the model quality and performance in molecular replacement with the Phaser software. 15 new cases of diffraction data sets that are unambiguously phased with de novo models are presented. These diffraction data sets represent nine space groups and span a large range of solvent contents (33-79%) and asymmetric unit copy numbers (1-4). No correlation is observed between the ease of phasing and the solvent content or asymmetric unit copy number. Instead, a weak correlation is found with the length of the modeled protein: larger proteins required somewhat less accurate models to give successful molecular replacement. Overall, the results of this survey suggest that de novo models can phase diffraction data for approximately one sixth of proteins with sizes of 100 residues or less. However, for many of these cases, ;de novo phasing with de novo models' requires significant investment of computational power, much greater than 10(3) CPU days per target. Improvements in conformational search methods will be necessary if molecular replacement with de novo models is to become a practical tool for targets without homology to previously solved protein structures.
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