Cell-free synthesis of amyloid fibrils with infectious properties and amenable to sub-milligram magic-angle spinning NMR analysis
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Published:2022-11-09
Issue:1
Volume:5
Page:
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ISSN:2399-3642
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Container-title:Communications Biology
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language:en
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Short-container-title:Commun Biol
Author:
Lends Alons, Daskalov Asen, Maleckis Ansis, Delamare Aline, Berbon Mélanie, Grélard Axelle, Morvan Estelle, Shenoy JayakrishnaORCID, Dutour Antoine, Tolchard James, Noubhani Abdelmajid, Giraud Marie-France, Sanchez Corinne, Habenstein Birgit, Guichard GillesORCID, Compain GuillaumeORCID, Jaudzems KristapsORCID, Saupe Sven J., Loquet AntoineORCID
Abstract
AbstractStructural investigations of amyloid fibrils often rely on heterologous bacterial overexpression of the protein of interest. Due to their inherent hydrophobicity and tendency to aggregate as inclusion bodies, many amyloid proteins are challenging to express in bacterial systems. Cell-free protein expression is a promising alternative to classical bacterial expression to produce hydrophobic proteins and introduce NMR-active isotopes that can improve and speed up the NMR analysis. Here we implement the cell-free synthesis of the functional amyloid prion HET-s(218-289). We present an interesting case where HET-s(218-289) directly assembles into infectious fibril in the cell-free expression mixture without the requirement of denaturation procedures and purification. By introducing tailored 13C and 15N isotopes or CF3 and 13CH2F labels at strategic amino-acid positions, we demonstrate that cell-free synthesized amyloid fibrils are readily amenable to high-resolution magic-angle spinning NMR at sub-milligram quantity.
Funder
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung Agence Nationale de la Recherche
Publisher
Springer Science and Business Media LLC
Subject
General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology,Medicine (miscellaneous)
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