Cell-free synthesis of amyloid fibrils with infectious properties and amenable to sub-milligram magic-angle spinning NMR analysis
Language
EN
Article de revue
This item was published in
Communications Biology. 2022-11-09, vol. 5, n° 1, p. 1-10
English Abstract
Structural 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 ...Read more >
Structural 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.Read less <
European Project
European Union (ERDF, INTERREGV)
ANR Project
Structure et fonctions de motifs amyloïdes impliqués dans la transduction du signal - ANR-17-CE11-0035
Interactions supramoléculaires sélectives et directionnelles à base de synthons fluorés hautement polaires
Interactions supramoléculaires sélectives et directionnelles à base de synthons fluorés hautement polaires