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Abstract:
Structural 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.
