ja504791j_si_001.pdf (1.42 MB)
Selective Methyl Labeling of Eukaryotic Membrane Proteins Using Cell-Free Expression
journal contribution
posted on 2015-12-17, 03:43 authored by Rasmus Linser, Vladimir Gelev, Franz Hagn, Haribabu Arthanari, Sven G. Hyberts, Gerhard WagnerStructural
characterization of membrane proteins and other large
proteins with NMR relies increasingly on perdeuteration combined with
incorporation of specifically protonated amino acid moieties, such
as methyl groups of isoleucines, valines, or leucines. The resulting
proton dilution reduces dipolar broadening producing sharper resonance
lines, ameliorates spectral crowding, and enables measuring of crucial
distances between and to methyl groups. While incorporation of specific
methyl labeling is now well established for bacterial expression using
suitable precursors, corresponding methods are still lacking for cell-free
expression, which is often the only choice for producing labeled eukaryotic
membrane proteins in mg quantities. Here we show that we can express
methyl-labeled human integral membrane proteins cost-effectively by
cell-free expression based of crude hydrolyzed ILV-labeled OmpX inclusion
bodies. These are obtained in Escherichia coli with very high quantity and represent an optimal intermediate to
channel ILV precursors into the eukaryotic proteins.