Wheat-germ cell-free production of prion proteins for solid-state NMR structural studies.
The expression of soluble, functional protein on a preparative scale poses a central challenge for structural studies. Cell-free protein expression has become a valuable alternative to cell-based methods, and allows today the expression of milligram quantities of protein. Its use is particularly attractive for NMR studies as it allows a multitude of isotopic labeling schemes. We have implemented and further developed protocols to prepare cell-free extracts from wheat germ to produce recombinant protein for solid-state NMR studies. Furthermore, we established the Renilla luciferase model to optimise and evaluate extract quality, and report first productions of the prions Ure2p and HET-s devoted to structural studies currently ongoing in our laboratories.
Noirot
Claire
C
Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS/Université de Lyon 1, 7 passage du Vercors, 69367 Lyon, France.
Habenstein
Birgit
B
Bousset
Luc
L
Melki
Ronald
R
Meier
Beat H
BH
Endo
Yaeta
Y
Penin
François
F
Böckmann
Anja
A
D000818:Animals; D002474:Cell-Free System / Q000378:metabolism*; D007553:Isotope Labeling; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011328:Prions / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014908:Triticum / Q000196:embryology*
D011328:Prions; D011994:Recombinant Proteins; D049410:Luciferases, Renilla