Robust Cell-Free Expression of Sub-Pathological and Pathological Huntingtin Exon-1 for NMR Studies. General Approaches for the Isotopic Labeling of Low-Complexity Proteins

Morató, Anna; Elena-Real, Carlos A.; Popovic, Matija; Fournet, Aurélie; Zhang, Karen; Allemand, Frédéric; Sibille, Nathalie; Urbanek, Annika; Bernadó, Pau

doi:10.3390/biom10101458

Cited by 11 publications

(14 citation statements)

References 94 publications

(172 reference statements)

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“…Cell-free systems (CFS) have recently revolutionized the field of synthetic biology by providing a fast, simple, and efficient platform for rapid genetic-circuit prototyping, biosensor engineering, and decentralized manufacturing . CFS can be used in basic research for production of recalcitrant or modified proteins, in educational kits, for building synthetic cells, , and for characterization of biological systems . They can be deployed for the prototyping of genetic circuits before in vivo implementation, , such as for metabolic engineering, , and also offer an attractive platform for engineering robust, stable, and regulatory compliant biosensors with applications in environmental monitoring and medical diagnostics. − …”

Section: Introductionmentioning

confidence: 99%

Differentially Optimized Cell-Free Buffer Enables Robust Expression from Unprotected Linear DNA in Exonuclease-Deficient Extracts

et al. 2022

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The use of linear DNA templates in cell-free systems promises to accelerate the prototyping and engineering of synthetic gene circuits. A key challenge is that linear templates are rapidly degraded by exonucleases present in cell extracts. Current approaches tackle the problem by adding exonuclease inhibitors and DNA-binding proteins to protect the linear DNA, requiring additional time-and resource-intensive steps. Here, we delete the recBCD exonuclease gene cluster from the Escherichia coli BL21 genome. We show that the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from σ70 promoters in linear DNA templates without employing additional protection strategies. When using linear or plasmid DNA templates at the buffer calibration step, the optimal potassium glutamate concentrations obtained when using linear DNA were consistently lower than those obtained when using plasmid DNA for the same extract. We demonstrate the robustness of the exonuclease deficient extracts across seven different batches and a wide range of experimental conditions across two different laboratories. Finally, we illustrate the use of the ΔrecBCD extracts for two applications: toehold switch characterization and enzyme screening. Our work provides a simple, efficient, and cost-effective solution for using linear DNA templates in cell-free systems and highlights the importance of specifically tailoring buffer composition for the final experimental setup. Our data also suggest that similar exonuclease deletion strategies can be applied to other species suitable for cell-free synthetic biology.

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Section: Introductionmentioning

confidence: 99%

Differentially Optimized Cell-Free Buffer Enables Robust Expression from Unprotected Linear DNA in Exonuclease-Deficient Extracts

et al. 2022

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“…The more details and protocol for preparation of S30 extract and other reagents are mentioned in the literature. − For labeling proteins, the labeled amino acid mixture at the concentration 0.3–2 mM is used. The labeled algal extracts are also used as the source of amino acids but lack Cys, Trp, Asn and Gln and these amino acids are required to be supplemented in the pure form . In CFPS, potassium l -glutamate is generally used as the source for K + ions but in case of uniform labeling, it creates a dilution effect for Glu residue.…”

Section: Sample Preparation (Labeling Strategies)mentioning

confidence: 99%

“…The labeled algal extracts are also used as the source of amino acids but lack Cys, Trp, Asn and Gln and these amino acids are required to be supplemented in the pure form. 105 In CFPS, potassium Lglutamate is generally used as the source for K + ions but in case of uniform labeling, it creates a dilution effect for Glu residue. The other sources of K + ions like potassium acetate and potassium D-glutamate are used to avoid these problems but result in lower yields.…”

Section: Uniform Labelingmentioning

confidence: 99%

Solid-State NMR: Methods for Biological Solids

et al. 2022

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In the last two decades, solid-state nuclear magnetic resonance (ssNMR) spectroscopy has transformed from a spectroscopic technique investigating small molecules and industrial polymers to a potent tool decrypting structure and underlying dynamics of complex biological systems, such as membrane proteins, fibrils, and assemblies, in near-physiological environments and temperatures. This transformation can be ascribed to improvements in hardware design, sample preparation, pulsed methods, isotope labeling strategies, resolution, and sensitivity. The fundamental engagement between nuclear spins and radio-frequency pulses in the presence of a strong static magnetic field is identical between solution and ssNMR, but the experimental procedures vastly differ because of the absence of molecular tumbling in solids. This review discusses routinely employed state-of-the-art static and MAS pulsed NMR methods relevant for biological samples with rotational correlation times exceeding 100’s of nanoseconds. Recent developments in signal filtering approaches, proton methodologies, and multiple acquisition techniques to boost sensitivity and speed up data acquisition at fast MAS are also discussed. Several examples of protein structures (globular, membrane, fibrils, and assemblies) solved with ssNMR spectroscopy have been considered. We also discuss integrated approaches to structurally characterize challenging biological systems and some newly emanating subdisciplines in ssNMR spectroscopy.

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“…By controlling the isotopic scheme of amino acids used in cell-free reactions, these approaches allow reducing isotopic scrambling. This strategy has been successfully applied for the NMR study of low-complexity regions of Htt exon1 combining cell-free expression using transcription-translation systems of Escherichia coli extracts and nonsense suppression for the site-specific isotopic labeling ( Morató et al, 2020 ).…”

Section: Deciphering the Posttranslational Modification Codes Of Amyl...mentioning

confidence: 99%

Deciphering the Structure and Formation of Amyloids in Neurodegenerative Diseases With Chemical Biology Tools

et al. 2022

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Protein aggregation into highly ordered, regularly repeated cross-β sheet structures called amyloid fibrils is closely associated to human disorders such as neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases, or systemic diseases like type II diabetes. Yet, in some cases, such as the HET-s prion, amyloids have biological functions. High-resolution structures of amyloids fibrils from cryo-electron microscopy have very recently highlighted their ultrastructural organization and polymorphisms. However, the molecular mechanisms and the role of co-factors (posttranslational modifications, non-proteinaceous components and other proteins) acting on the fibril formation are still poorly understood. Whether amyloid fibrils play a toxic or protective role in the pathogenesis of neurodegenerative diseases remains to be elucidated. Furthermore, such aberrant protein-protein interactions challenge the search of small-molecule drugs or immunotherapy approaches targeting amyloid formation. In this review, we describe how chemical biology tools contribute to new insights on the mode of action of amyloidogenic proteins and peptides, defining their structural signature and aggregation pathways by capturing their molecular details and conformational heterogeneity. Challenging the imagination of scientists, this constantly expanding field provides crucial tools to unravel mechanistic detail of amyloid formation such as semisynthetic proteins and small-molecule sensors of conformational changes and/or aggregation. Protein engineering methods and bioorthogonal chemistry for the introduction of protein chemical modifications are additional fruitful strategies to tackle the challenge of understanding amyloid formation.

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Robust Cell-Free Expression of Sub-Pathological and Pathological Huntingtin Exon-1 for NMR Studies. General Approaches for the Isotopic Labeling of Low-Complexity Proteins

Cited by 11 publications

References 94 publications

Differentially Optimized Cell-Free Buffer Enables Robust Expression from Unprotected Linear DNA in Exonuclease-Deficient Extracts

Differentially Optimized Cell-Free Buffer Enables Robust Expression from Unprotected Linear DNA in Exonuclease-Deficient Extracts

Solid-State NMR: Methods for Biological Solids

Deciphering the Structure and Formation of Amyloids in Neurodegenerative Diseases With Chemical Biology Tools

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