Abstract
The cell-free synthesis is an efficient strategy to produce in large scale protein samples for structural investigations. In vitro synthesis allows for significant reduction of production time, simplification of purification steps and enables production of both soluble and membrane proteins. The cell-free reaction is an open system and can be performed in presence of many additives such as cofactors, inhibitors, redox systems, chaperones, detergents, lipids, nanodisks, and surfactants to allow for the expression of toxic membrane proteins or intrinsically disordered proteins. In this chapter we present protocols to prepare E. coli S30 cellular extracts, T7 RNA polymerase, and their use for in vitro protein expression. Optimizations of the protocol are presented for preparation of protein samples enriched in deuterium, a prerequisite for the study of high-molecular-weight proteins by NMR spectroscopy. An efficient production of perdeuterated proteins is achieved together with a full protonation of all the amide NMR probes, without suffering from residual protonation on aliphatic carbons. Application to the production of the 468 kDa TET2 protein assembly for NMR investigations is presented.
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Acknowledgments
The authors thank Prof. Eva Pebay-Peyroula for providing the clone of the HMA8 ATPases nucleotide-binding domain from Arabidopsis thaliana. This work used the high field NMR and Cell-Free facilities at the Grenoble Instruct-ERIC Center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB). Platform access was supported by FRISBI (ANR-10-INBS-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA). This work was supported by grants from CEA/NMR-Bio (research program C24990) and the Agence Nationale de la Recherche (ANR-17-CE29-0010 CH2-PROBE).
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Imbert, L. et al. (2021). In Vitro Production of Perdeuterated Proteins in H2O for Biomolecular NMR Studies. In: Chen, Y.W., Yiu, CP.B. (eds) Structural Genomics. Methods in Molecular Biology, vol 2199. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0892-0_8
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