Wheat-germ cell-free production of prion proteins for solid-state NMR structural studies

Noirot, Claire; Habenstein, Birgit; Bousset, Luc; Melki, Ronald; Meier, Beat H.; Endo, Yaeta; Pénin, François; Böckmann, Anja

doi:10.1016/j.nbt.2010.06.016

Cited by 7 publications

(3 citation statements)

References 46 publications

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“…In short, home-made wheat germ extracts (Noirot et al 2011) were prepared according to Takai et al (Takai et al 2010 Transcription and translation were carried out separately, as described in (Takai et al 2010) and (Noirot et al 2011), and translation was performed using the so-called bilayer method.…”

Section: Wheat Germ Cell-free Protein Expressionmentioning

confidence: 99%

“…In that case, cell-free production is an interesting alternative (Betton 2004;Vinarov et al 2004;Klammt et al 2012), as in particular it offers the opportunity for complex labeling schemes for NMR (Kainosho et al 2006;Tonelli et al 2011). While cell-free expression using E. coli extracts remains the most widely used and successful approach, an interesting alternative for eukaryotic proteins is the wheat germ cell-free expression system (Sawasaki et al 2002;Endo and Sawasaki 2006;Takai et al 2010;Noirot et al 2011;Fogeron et al 2015a). Indeed, it combines the advantages of cell-free expression with the benefits of the slow translation rates inherent to eukaryotic ribosomes, thus being more apt in supporting adequate folding, although it has been shown that also in E. Coli lysates the translation rates are slower than in vivo.…”

Section: Introductionmentioning

confidence: 99%

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Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

et al. 2016

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Section: Wheat Germ Cell-free Protein Expressionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

et al. 2016

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“…In wheat germ extracts metabolic activities are commonly low or can be inactivated, and the system is successfully used for effective preparation of labeled proteins in protein NMR studies [98][99][100] yielding very high incorporation rates. Furthermore, a method for incorporation of deuterated amino acids has been described [101], and the approach has been successfully applied to solid-state NMR studies [102]. Also the larger protein requirements for X-ray crystallography can be addressed by the wheat germ system, where automated protein synthesizers are useful devices to prepare multi milligram amounts of high quality protein [36,39] with the possibility to incorporate selenomethionine.…”

Section: Structural Genomics Nmr and Crystallographymentioning

confidence: 99%

Wheat germ systems for cell‐free protein expression

Harbers

2014

FEBS Letters

101

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a b s t r a c tCell-free protein expression plays an important role in biochemical research. However, only recent developments led to new methods to rapidly synthesize preparative amounts of protein that make cell-free protein expression an attractive alternative to cell-based methods. In particular the wheat germ system provides the highest translation efficiency among eukaryotic cell-free protein expression approaches and has a very high success rate for the expression of soluble proteins of good quality. As an open in vitro method, the wheat germ system is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Here I describe wheat germ cellfree protein expression systems and give examples how they have been used in genome-wide expression studies, preparation of labeled proteins for structural genomics and protein mass spectroscopy, automated protein synthesis, and screening of enzymatic activities. Future directions for the use of cell-free expression methods are discussed.

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How to Investigate Interactions Between Membrane Proteins and Ligands by Solid-State NMR

Lakatos

Mörs

Glaubitz

2012

Methods in Molecular Biology

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Solid-state NMR is an established method for biophysical studies of membrane proteins within the lipid bilayers and an emerging technique for structural biology in general. In particular magic angle sample spinning has been found to be very useful for the investigation of large membrane proteins and their interaction with small molecules within the lipid bilayer. Using a number of examples, we illustrate and discuss in this chapter, which information can be gained and which experimental parameters need to be considered when planning such experiments. We focus especially on the interaction of diffusive ligands with membrane proteins.

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Wheat-germ cell-free production of prion proteins for solid-state NMR structural studies

Cited by 7 publications

References 46 publications

Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

Wheat germ systems for cell‐free protein expression

How to Investigate Interactions Between Membrane Proteins and Ligands by Solid-State NMR

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